Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants

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Abstract
RNA-mediated virus resistance has recently been shown to be the result of post-transcriptional transgene silencing in transgenic plants. This study was undertaken to characterize the effect of transgene length and nontarget DNA sequences on RNA-mediated tospovirus resistance in transgenic plants. Transgenic Nicotiana benthamiana plants were generated to express different regions of the nucleocapsid (N) protein of tomato spotted wilt (TSWV) tospovirus. Transgenic plants expressing half-gene segments (387–453 bp) of the N gene displayed resistance through post-transcriptional gene silencing. Although smaller N gene segments (92–235 bp) were ineffective in conferring resistance when expressed alone in transgenic plants, these segments conferred resistance when fused to the nontarget green fluorescent protein gene DNA. These results demonstrate that (i) a critical length of N transgene (236–387 bp) is required for a high level of transgene expression and consequent gene silencing, and (ii) the post-transcriptional gene silencing mechanism can trans-inactivate the incoming tospovirus genome with homologous transgene segments that are as short as 110 bp. Therefore, the activation of post-transcriptional transgene silencing requires a significantly larger transgene than is required for the trans-inactivation of the incoming viral genome. These results raise the possibility of developing a simple new strategy for engineering multiple virus resistance in transgenic plants.