Ribozyme Mediated Targeting of Cucumber Mosaic Virus RNA 1 and 2 in Transgenic Tobacco Plants

Hammerhead ribozymes have been extensively used to inhibit the expression of cellular genes or viral genes mainly in the animal study. In this study, we designed a ribozyme targeting the conserved leader sequences of cucumber mosaic virus (CMV) RNA 1 and 2. The ribozyme, with asymmetric lengths of flanking complementary regions, cleaved a model substrate RNA efficiently at 26 °C as well as at 37 °C or 50 °C in vitro. And the ribozyme encoding sequence was introduced into tobacco plants and expressed with the CaMV 35S promoter and 3′ NOS terminator in a monomeric type (pBIR1), tandemly repeated type (pBIR3), and cotranscriptionally combined type (pRokR) with 2.2 copies of I₁₇N satellite RNA. Virus challenging experiments in F₁ plants of respective transformants with CMV-Y showed specific reductions of viral RNA 1 and 2 in comparison with RNA 3 or 4. Although young plants of a three-leaf-stage showed rather similar mild symptom attenuations in all constructions compared to CMV-Y inoculated wild type, fully grown plants showed a differential degree of resistance upon systemic infections of CMV-Y in pRokK, pBIR3 and pBIR1 transformed plants in a decreasing order.