Transgenic resistance to tomato spotted wilt virus in tobacco lines transformed with the nucleocapsid gene of the virus: Field assessment and molecular characterization

In the first part of this doctoral thesis, we developed transgenic tobacco lines resistant to tomato spotted wilt virus (TSWV), and after extensive greenhouse evaluation, we assessed the spectrum of transgenic resistance in tobacco to naturally occurring TSWV strains. We transformed three different tobacco cultivars with the N gene of a dahlia isolate of TSWV (TSWV-D). Greenhouse evaluation with TSWV-D and selection of resistant lines over several generations indicated that expression of the resistant phenotype is more stable in cv. Burley 21 than in cvs. KY-14 or K-326. Three transgenic ‘Burley 21’ lines were identified which had significantly lower incidence of disease than susceptible controls over the two years of the study. We conclude that lines which show the highest levels of resistance to natural inoculum may be those that express high levels of transgene protein. Alternatively, N protein accumulation in the field-grown plants may indicate latent infection, suggesting that transgenic plants require a higher level of virus accumulation for symptom expression.; In the second part of this work, we developed five segregating tobacco families transformed with N gene of TSWV-D, and we used these to investigate the correlation between the expression of resistance and its molecular determinants such as transgene dosage, transcript and/or protein accumulation. The results suggest that transgene dosage is directly correlated with the expression of resistance. Conversely, the other two families transformed with the sense construct showed no correlation between the number of insertions and the resistance response. However, 82% of the resistant plants had a common 8.0Kb insertion that was actively transcribed, while 77% of the susceptible plants had an insertion that was not transcribed as efficiently. Transgene protein accumulation was not detected in any of the plants analyzed, indicating that resistance to TSVW in the transgenic tobacco families analyzed is most likely RNA-mediated. (Abstract shortened by UMI.)...