RNA interference (RNAI) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes. In this study a microRNA 159a-based binary vector was constructed which can be used for hpRNA expression. Hairpin (hp) RNA expression cassettes carrying the gene sequences are typically constructed on binary plasmid and delivered into plant cells by Agrobacteriurn-mediated genetic transformation. This system allows simple insertion of 21- nt target gene sequences into microRNA backbone, to facilitate the processing of microRNA hpRNA by the endogenous machinery of host, thereby producing artificial microRNA carrying the sequence of target gene(s). The functionality of new vector system was tested by silencing viral gene in transgenic plants. Strong down regulation of viral gene was observed in virus infected tobacco plants transformed with PAmiR(159) vector. The processing of amiRNA leading to viral-specific siRNA was confirmed by northern blotting. This vector system provides an important addition to the plant molecular biologists' toolbox, which will significantly facilitate the use of RNAi technology for analyses of various gene functions in plant cells.