Tomato leaf curl virus satellite DNA as a gene silencing vector activated by helper virus infection

Tomato leaf curl virus (TLCV) satellite DNA (sat-DNA) constructs containing functional segments of the cauliflower mosaic virus (CaMV) 35S promoter, replicate in tobacco in the presence of helper TLCV and silence GUS activity in transgenic tobacco plants containing a CaMV 35S-GUS expression cassette. We have analysed these plants for evidence of the hallmarks of silencing. The GUS transcript was not detectable in the leaves of GUS-silenced tobacco plants. These plants contained siRNAs of approximately 23 nt in length homologous to both the 35S promoter region and the GUS ORF. The absence of GUS expression and the existence of siRNAs in transgenic plants show that the silencing induced by TLCV sat-DNA is due to RNA silencing. To test the utility of this silencing system, a 341 nucleotide promoter sequence of the petunia chalcone synthase A (ChsA) was inserted into the sat-DNA and inoculated into petunia plants, together with the helper TLCV, and found to markedly reduce pigmentation of flowers and the level of ChsA transcript. This DNA-based silencing system has the potential to introduce epigenetic traits via short DNA inserts to a variety of plants that are hosts to different geminiviruses supporting the sat-DNA.