Ribozyme-mediated resistance to rice dwarf virus and the transgene silencing in the progeny of transgenic rice plants.

A hammerhead ribozyme (Rz) with long hybridizing arms targeting the mRNA of rice dwarf virus (RDV) segment 5 and a mutated nonfunctional ribozyme (mRz) were constructed. As predicted, Rz transcribed in vitro cleaved the target mRNA of RDV segment 5 into two fragments of 138 and 238 nucleotides in length. The Rz and mRz genes were each placed under the control of the CaMV 35S promoter and used to transform Japonica rice variety 'Tongling No. 1' via Agrobacterium tumefaciens. A total of 32 independent lines containing Rz or mRz was obtained as demonstrated by Southern blot analysis. Challenge inoculation with RDV viruliferous leafhoppers (Nephotettix cincticeps) showed that T1 plants containing the Rz transgene displayed high resistance or delayed and attenuated viral symptoms. In contrast, transgenic lines expressing mRz showed severe symptoms similar to the control plants transformed with the vector alone. These results suggest that Rz confers RDV resistance in transgenic rice. Genomic DNA PCR analysis confirmed that all of the examined T6 progeny plants contained the Rz transgene. However, accumulation of the Rz transcripts was detectable by RT-PCR only in the plants that were resistant to RDV. This suggested that loss of RDV resistance in progeny plants containing the Rz transgene may result from silencing of the Rz transgene.