Analysis Of The Resistance Mediated By RNA Silencing In Transgenic Tobacco Plants

Recently, RNA-silencing mediated resistance is being widely used to generate virus-resistant transgenic plants. RNA silencing provides plants with a high level of resistance by specific targeting to cognate viral RNA. The discovery of unstable resistance at T4-T5 progeny of transgenic high-resistant plants questions the stability of RNA-silencing mediated resistance. Moreover, observation of RNA silencing inhibition by virus encoded suppressors or low temperature environment also leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRP) has been previously characterized to be essential for transgene-mediated RNA silencing and the efficiency of this antiviral activity is tightly linked to temperature. Here we investigated the stability of transgenic resistance in T4 progeny transgenic high-resistant line M11 plants. The main results are as follows:1. Through virus-challenging experiment, we proved that the high resistance of line M11 plants could be transmitted steadily to T4 progeny. Northern blot assays revealed that the transgene transcriptional level was inversely correlated to the resistance, which means the resistance was mediated by RNA-silencing.2. NtRdRP1 was involved in the RNA-silencing. We cloned a 995bp-fragment as probe, and subsequently conducted Northern blot assays to transgenic plants. The results showed that the silencing M11 plants accumulated much higher NtRdRP1 level than the control plants. This provides powerful evidence to support the involvement of NtRdRP1 in RNA silencing.3. Conducting virus-challenge experiments at alterative temperatures revealed that low temperature inhibited the RNA-silencing mediated resistance, and this was further confirmed on molecular level by Northern blot assays.4. Northern blot assays indicated that the NtRdRP1 expression is temperature-dependent, which reduced at low temperature and increased with temperature rising. This supports the belief that NtRdRP1 involved in the RNA silencing.5. RNA silencing was differently suppressed by PVX and TMV infection, whereas both resulting in the disruption of resistance in transgenic plants. PVX infection alleviated the RNA silencing of transgenic plants in newly emerged leaves, other than in old leaves, whereas the TMV inhibited the RNA silencing of transgenic plants in both old and newly emerged leaves.6. Our results showed that NtRdRP1 expression was elevated in both the old and the newly emerged leaves post either PVX or TMV infection. The similar pattern induced by the two unrelated viruses implied that this response to viruses would be a general feature of NtRdRP1.