Characteristics of Resistance to Potato Virus Y in Transgenic Tobacco Plants Mediated with Complimentary DNA (cDNA) of PVY Replicase Mutant Genes

  • Published : 1998.06.01

Abstract

This study was conducted to develop a resistant tobarro against Potato virus Y (PVY) by transformation of the plants with genetically engineered viral genes. The complimentary DNAs (cDNAS) of potato virus Y-necrosis strain (PVY-Vn) replicase mutant genes (3'-deleted, 5'-deleted and ADD-mutant Nlbs) were synthesized through RT-PCR by using purified PVY-VN RNA and synthesized primers, and cloned in the sense orientation into a plant expression vector (pMBPI), The cDNAS of the genes were transferred into Agrobacterium tumefaciens LBA 4404, and then transformed into tobacco (Nicotiana tabacum cv. Burley 21) plants. Regenerated plants were tested for PVY resistance by inoculation test; 13 transgenic plants including 7 for 3'-deleted Nlb, 3 for 5'-deleted Nlb, and 3 for ADD-mutant Nlb appeared to be resistant at 4 weeks after inoculation with PVY-VN. Among the 13 transgenic tobacco plants, 8 plants had no symptom up to 14 weeks after inoculation. The progenies ($T_1$) from self-fertilization of the transgenic lines varied 0.0% to 81.2% in their resistance (% of resistant plants). The analysis of Nlb-31deleted, -5'deleted and -ADD mutant in the $T_1$ plants by polymerase chain reaction (PCR) showed that Nlb-3'deleted, -5'deleted and -ADD mutants were detected in all of the resistant plants. These results suggest that the PVY resistance was inherited in the $T_1$ generation.

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References

  1. Methods Enzymol v.153 Binary Ti vectors for plant transformation and promoter analysis An, G.
  2. Plant Cell v.8 Mechanisms of pathogenderived resistance to viruses in transgenic plants Baulcombe, D. C.
  3. Annu. Rev. Phytopathol v.28 Coat protein mediated resistance against virus infection Beachy, R. N.;S. Loesch-Fries;N. E. Tumer
  4. Gene v.19 Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transponson Tn5 Beck, E.;G. Ludwig;E. A. Averswald;B. Reiss;H. Schaller
  5. Mol. Cells v.4 Isolation and Nucleotide sequence analysis of a patial cDNA clone for potato virus Y-VN (Korean isolate) genome Cho, H. S.;S. I. Jun;S. Y. Kwon;E. K. Park;K. Y. Paek;K. H. Paek
  6. J. Gen. Virol v.34 Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses Clack, M. F.;A. N. Adams
  7. Nucleic Acids Res v.19 A simple and rapid method for the preparation of plant genomic DNA for PCR analysis Edwards, K.;C. Johnstone;C. Thompson
  8. Science v.227 A simple and general method for transferring genes into plants Horsch, R. B.;J. E. Fry;N. L. Hoffman;D. Eichholtz;S. G. Rogers;R. T. Fraley
  9. Mol. Plant-Micro. Interact v.5 Pathogen-dericed resistance to potyvirus: Immune and resistance phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence Lindbo, J. A.;G. W. Dougherty
  10. Disease of Tobacco (Thirf Ed.) Biological Consultants Associates Lucas, G. B.
  11. Physiol. Plant v.15 A revised medium for rapid growth and bioassays with tobacco tissue culture Murashige, T.;F. Skoog
  12. J. Gen. Virol v.70 Nucleotide sequence of potato virus Y (N strain) genomic RNA Robaglia, C.;M. Durand-Tardif;M. Tronchet;G. Boudazin;S. Astier-Manifacier;F. CasseDelbart
  13. Proc. Natl. Acad. Sci. USA v.74 DNA sequencing with chain-terminating inhibitors Sanger, F.;S. Nicklen;A. R. Coulson