| Abstract |
In plants, the expression of double-stranded RNA causes RNA silencing which can be used specifically to downregulate any target gene. This technology has been successfully applied to engineer virus-resistant plants. However, under field conditions host plants may be infected by different viruses. Multiple infections may cause severe disease symptoms. We intended to generate transgenic tobacco plants that are concurrently resistant to three different RNA viruses: tomato spotted wilt tospovirus (TSWV), cucumber mosaic cucumovirus (CMV) and potato virus Y (PVY). For that purpose we followed two experimental strategies. First, we cloned cDNA fragments of each of the three viruses that are conserved in sequence in different isolates. These cDNA fragments were combined to a single recombinant unit and subcloned to a plasmid vector in front of a cDNA encoding an intron of glutamate dehydrogenase (GDH) from Vitis vinifera. A second copy of the tripartite cDNA unit was inserted downstream of the intron sequence, however, in inverted orientation. The combined cDNA was brought under the control of the CaMV35S promoter. After transcription and excision of the intron, the RNA molecule will form, theoretically, a double-stranded molecule with regions specific for each of the three viruses. Second, we tried to simplify this approach by ligating chemically synthesized DNA fragments to a single recombinant cDNA, specific for all three viruses. The construction of the actual hairpin followed the same strategy. The usage of chemically synthesized DNA fragments requires only sequence information without the need of cloning the viral cDNA. Both constructs were used for the transformation of tobacco plants of the oriental variety Basmas N34-4.
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