Post-transcriptional silencing of chalcone synthase in petunia using a geminivirus-based episomal vector

A vector that produces DNA replicons (multicopy plant episomes) was constructed using elements of the geminivirus tobacco yellow dwarf virus (TYDV). All plant cells contain an integrated chromosomal T-DNA copy of the TYDV elements that provides a template for the production of episomes in the cell nucleus. Transgenic Petunia hybrida plants containing a CaMV 35S promoter-driven chalcone synthase A (ChsA) gene cloned into the episomal vector produced flowers with a white-spotted phenotype at high frequency. The spots were found at random locations in the petals and occurred in corresponding positions in both the upper and lower epidermis, indicating that the spots were non-clonal. The spotted phenotype was somatically stable and was inherited through meiosis. In white-spotted flower tissue, steady-state ChsA mRNA levels were downregulated but rates of RNA transcription were unaffected, suggesting that the phenotype resulted from post-transcriptional gene silencing of the endogenous and episomal ChsA genes. Increases in both the frequency and extent of gene silencing in flowers correlated with increases in episome copy number in mature flowers, flower buds and young and fully expanded leaves. Relatively small increases in episome copy number (less than threefold) appeared sufficient to trigger the gene-silenced phenotype.