Transgenic tobacco lines expressing defective CMV replicase-derived dsRNA are resistant to CMV-O and CMV-Y.

Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus causing infections and yield losses to many plant species. Here, we generated a construct containing inverted repeat of 1,793 bp fragment of defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). The replicase gene was modified by deleting a 9 bp region between nucleotides 1909-1918. This caused a deletion in the active centre motif of polymerases, producing defective translated product 9 nucleotides shorter than the full length protein. The RNAi construct containing inverted repeat of the defective gene was used to produce transgenic tobacco lines expressing CMV-derived double-stranded RNA via Agrobacterium-mediated transformation. Of the four transgenic lines inoculated with CMV-O or CMV-Y in vitro and ex vivo, three lines (T1, T4 and T5) showed immunity to both strains of CMV as no symptoms were detected, whereas one line (T7) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and Dot-immunobinding assay analyses. Small interfering RNAs present in transgenic lines before and after virus challenge indicates that the resistance was acquired through RNA silencing.