The instability of the inhibition of the expression of genes involved in resistance to virus infection in transgenic plants using hairpin constructs

Transgenic expression of hairpins can induce RNA silencing pathways by means of dsRNA with sequence homology to a plant mRNA. Using transgenic tobacco plants (N. tabacum cv. Samsun NN) in which the RDR1 gene was silenced to different extents by a hairpin structure homologous to the tobacco RDR1 gene، we evaluated the stability of gene silencing، by analyzing the RDR1 gene expression levels and the plant susceptibility to systemic infection by PVY°. The second (T2) and third (T3) generations of RDR1 silenced transgenic lines were tested by semi-quantitative RT-PCR to evaluate the expression levels of defense-related genes including IVR، ERF5، AOX1، and RDR6، one week after PVY° infection. The results showed that T2 transgenic tobacco lines transcribed the RDR1 and the other defense-related genes to a lower level than the corresponding T3 transgenic lines. The T2 lines were more susceptible to PVY° infection than the T3 lines and the RDR1 gene was expressed to the same level in the T3 lines as in non-transformed tobacco. The efficiency of hairpin structure-mediated silencing of the RDR1 gene decreased through different generations. As the RDR1-regulated RDR6 is required for the spreading of silencing throughout the plant، we propose that suppressing RDR1 leads to inhibition of RDR6، which then leads to loss of silencing by the RDR1 hairpin transgene after vertical gene transmission.