Analyzing the impact of transgene-derived siRNAs on tomato yellow leaf curl Sardinia virus infection.

We have recently shown that tomato yellow leaf curl Sardinia virus (TYLCSV) is able to overcome Rep-210-mediated resistance by shutting-off transgene expression by a posttranscriptional homology-dependent mechanism, suggesting that TYLCSV should be able to evade at some extent transgene-mediated silencing of the essential viral Rep gene (Lucioli et al. 2003 J. Virol. 77:6785). In this report we directly analysed the impact of silencing on TYLCSV infection, using two classes of posttranscriptionally silenced Rep-210 transgenic tomato plants. One class consisted of sense x antisense Rep-210 hybrid while the other was the selfed-progeny of tomato lines that integrated multiple copies of the sense Rep-210 transgene. In both classes, posttranscriptionally silenced plants accumulated low or undetectable amount of Rep-210 protein and mRNA, but high amount of Rep-210 transgene-specific siRNAs. Interestingly, all silenced plants were susceptible to TYLCSV when challenged by agroinoculation. However, when these plants were challenged with viruliferous Bemisia tabaci, the ratio between susceptible versus resistant plants was dependent on the inoculum load. At high viral vector inoculum, all the silenced plants were susceptible. Interestingly, and according to the dose effect, some plants showed delayed infection, indicating that TYLCSV exerts an active role in overcoming transgene-derived siRNA-targeting of its Rep gene. The overall data suggest a threshold model in which transgene-derived siRNAs interfere with virus infection in the initially inoculated cells but, if viral replication/expression reaches a threshold, virus spreading in the silenced tissue cannot be any more prevented