1887

Abstract

Three transgenic lines of squash hemizygous for the coat protein genes of squash mosaic virus (SqMV) were shown previously to have resistant (SqMV-127), susceptible (SqMV-22) or recovery (SqMV-3) phenotypes. Post-transcriptional gene silencing (PTGS) was the underlying mechanism for resistance of SqMV-127. Here, experiments conducted to determine the mechanism of the recovery phenotype and whether enhanced resistance could be obtained by combining transgenes from susceptible and recovery plants are reported. Upper leaves of SqMV-3 plants were sampled for Northern analysis at 17, 31 and 45 days after germination (DAG) and a proportion of plants were inoculated with SqMV. SqMV-3 plants inoculated at a young stage (17 DAG) showed susceptible or recovery phenotypes. However, a number of plants inoculated at later developmental stages (31 or 45 DAG) were resistant to infection. Resistance of recovery plants was due to PTGS that was activated at a later developmental stage, independent of virus infection. Similar results were observed with plants grown under field conditions. To investigate the interactions of transgenes, progeny of crosses between SqMV-127, -3 and -22 were inoculated with SqMV. Progeny with the transgene of line 127 were resistant. However, a number of plants with transgenes from the recovery and susceptible lines or the self-pollinated recovery line were resistant even when inoculated at a young stage. Northern analysis suggested that resistance was due to PTGS. The results reveal that the timing of PTGS and consequent resistance of the transgenic plants were affected by their developmental stage and the interaction of transgene inserts.

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2000-09-01
2024-05-11
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References

  1. Balandin T., Castresana C. 1997; Silencing of a β-1,3-glucanase transgene is overcome during seed formation. Plant Molecular Biology 34:125–137
    [Google Scholar]
  2. Bass B. L. 2000; Double-stranded RNA as a template for gene silencing. Cell 101:235–238
    [Google Scholar]
  3. Baulcombe D. C. 1996; Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8:1833–1844
    [Google Scholar]
  4. Baulcombe D. C., English J. J. 1996; Ectopic pairing of homologous DNA and post-transcriptional gene silencing in transgenic plants. Current Opinion in Biotechnology 7:173–180
    [Google Scholar]
  5. Beachy R. N. 1997; Mechanisms and applications of pathogen-derived resistance in transgenic plants. Current Opinion in Biotechnology 8:215–220
    [Google Scholar]
  6. Campbell R. N. 1971; Squash mosaic virus . . CMI/AAB Descriptions of Plant Viruses no. 43
    [Google Scholar]
  7. Clark M. F., Adams A. N. 1977; Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34:475–483
    [Google Scholar]
  8. Dawson W. D. 1996; Gene silencing and virus resistance, a common mechanism. Trends in Plant Science 1:107–108
    [Google Scholar]
  9. Dehio C., Schell J. 1994; Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing. Proceedings of the National Academy of Sciences, USA 91:5538–5542
    [Google Scholar]
  10. Dougherty W. G., Parks T. D. 1995; Transgenes and gene suppression: telling us something new?. Current Opinion in Cell Biology 7:399–405
    [Google Scholar]
  11. Doyle J. J., Doyle J. L. 1990; Isolation of plant DNA from fresh tissue. Focus 12:13–15
    [Google Scholar]
  12. English J. J., Mueller E., Baulcombe D. C. 1996; Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8:179–188
    [Google Scholar]
  13. Feinberg A. P., Vogelstein B. 1983; A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132:6–13
    [Google Scholar]
  14. Franssen H., Goldbach R., Broekhuijsen M., Moerman M., van Kammen A. 1982; Expression of middle-component RNA of cowpea mosaic virus, in vitro generation of a precursor to both capsid proteins by bottom-component RNA-encoded protease from infected cells. Journal of Virology 41:8–17
    [Google Scholar]
  15. Goldbach R. W., Wellink J. 1996; Comoviruses, molecular biology and replication. In The Plant Viruses pp 35–76 Edited by Harrison B. D., Murant A. F. New York: Plenum Press;
    [Google Scholar]
  16. Goodwin J., Chapman K., Swaney S., Parks T. D., Wernsman E. A., Dougherty W. G. 1996; Genetic and biochemical dissection of transgenic RNA-mediated virus resistance. Plant Cell 8:95–105
    [Google Scholar]
  17. Guo H. S., García 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
    [Google Scholar]
  18. Hamilton A. J., Baulcombe D. C. 1999; A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952
    [Google Scholar]
  19. Haudenshield J. S., Palukaitis P. 1998; Diversity among isolates of squash mosaic virus. Journal of General Virology 79:2331–2341
    [Google Scholar]
  20. Hu J. S., Pang S.-Z., Nagpala P. G., Siemieniak D. R., Slightom J. L., Gonsalves D. 1993; The coat protein genes of squash mosaic virus: cloning, sequence analysis, and expression in tobacco protoplasts. Archives of Virology 130:17–31
    [Google Scholar]
  21. Kunz C., Schob H., Stam M., Kooter J. M., Meins F. J. 1996; Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression. Plant Journal 10:437–450
    [Google Scholar]
  22. Lindbo J. A., Silva R. 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
    [Google Scholar]
  23. Lomonossoff G. P., Shanks M. 1983; The nucleotide sequence of cowpea mosaic virus B RNA. EMBO Journal 2:2253–2258
    [Google Scholar]
  24. Metzlaff M., O’Dell M., Cluster P. D., Flavell R. B. 1997; RNA-mediated RNA degradation and chalcone synthase A silencing in petunia. Cell 88:845–854
    [Google Scholar]
  25. Montgomery M. K., Fire A. 1998; Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends in Genetics 14:255–258
    [Google Scholar]
  26. Mueller E., Gilbert J., Davenport G., Brigneti G., Baulcombe D. C. 1995; Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing. Plant Journal 7:1001–1013
    [Google Scholar]
  27. 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 2:279–290
    [Google Scholar]
  28. Nelson M. R., Knuhtsen H. K. 1973; Squash mosaic virus variability: review and serological comparisons of six biotypes. Phytopathology 63:920–926
    [Google Scholar]
  29. Pang S.-Z., Jan F.-J., Carney K., Stout J., Tricoli D. M., Quemada H. D., Gonsalves D. 1996; Post-transcriptional transgene silencing and consequent tospovirus resistance in transgenic lettuce are affected by transgene dosage and plant development. Plant Journal 9:899–909
    [Google Scholar]
  30. 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
    [Google Scholar]
  31. Pang S.-Z., Jan F.-J., Tricoli D. M., Russell P. F., Carney K. J., Hu J. S., Fuchs M., Quemada H. D., Gonsalves D. 2000; Resistance to squash mosaic comovirus in transgenic squash plants expressing its coat protein genes. Molecular Breeding 6:87–93
    [Google Scholar]
  32. Prins M., Goldbach R. 1996; RNA-mediated virus resistance in transgenic plants. Archives of Virology 141:2259–2276
    [Google Scholar]
  33. Provvidenti R. 1993; Resistance to viral diseases of cucurbits. In Resistance to Viral Diseases of Vegetables pp 8–43 Edited by Kyle M. M. Portland, OR: Timber Press;
    [Google Scholar]
  34. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  35. Schiebel W., Pelissier T., Riedel L., Thalmeir S., Schiebel R., Kempe D., Lottspeich F., Sanger H. L., Wassenegger M. 1998; Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato. Plant Cell 10:2087–2101
    [Google Scholar]
  36. Smith H. A., Swaney S. L., Parks T. D., Wernsman E. A., Dougherty W. G. 1994; Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAs. Plant Cell 6:1441–1453
    [Google Scholar]
  37. 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
    [Google Scholar]
  38. Vaucheret H., Palauqui J. C., Elmayan T., Moffatt B. 1995; Molecular and genetic analysis of nitrite reductase co-suppression in transgenic tobacco plants. Molecular & General Genetics 248:311–317
    [Google Scholar]
  39. Waterhouse P. M., Graham M. W., Wang M. B. 1998; Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences, USA 95:13959–13964
    [Google Scholar]
  40. Wellink J., van Kammen A. 1989; Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins. Journal of General Virology 70:2279–2286
    [Google Scholar]
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