Spreading of post-transcriptional gene silencing along the target gene promotes systemic silencing

Transitive silencing and grafting-induced gene silencing phenomena were combined to investigate whether a primary target beta-glucuronidase (gus) gene could promote the generation of systemic transitive silencing signals. Tobacco plants with hemizygous or homozygous silencer locus and in trans silenced primary target were used as a source of post-transcriptionally silenced rootstocks and tobacco plants with or without a secondary target locus as scion source. The silencer locus harbored two identical neomycin phosphotransferase II (nptII)-containing T-DNAs, integrated as an inverted repeat. The primary target locus carried a gus gene with homology to the transcribed region of the nptII gene only in the 3' untranslated region, whereas the secondary target locus had two or more copies of a gus gene without homology to transcribed sequences of the silencer locus. The upstream region of the initially targeted sequences of the in trans silenced gus gene could induce the production of a systemic signal. This signal was capable of triggering post-transcriptional gene silencing (PTGS) of the secondary target gus genes in the scion. In addition, the induction of systemic silencing was strikingly dosage dependent for the silencer as well as the primary target loci in the rootstock. Moreover, in the scions, the secondary target gus genes had to be present to generate detectable amounts of short interfering RNAs.