Effect of orientation of transcription of a gene in an inverted transferred DNA repeat on transcriptional gene silencing in rice transgenics-a case study.

We studied transgene silencing in two transgenic rice plants, OSM25 and COT-OSM4, which harboured two different types of right border (RB)-centered inverted transferred DNA (T-DNA) repeats (IRs). The T-DNA in OSM25 has three genes gus, OSM and hph, all under the transcriptional control of the Cauliflower mosaic virus 35S promoter (P35S). The gus gene, which is proximal to the RB, is in a convergent orientation of transcription in the IR. OSM25 displayed silencing of all three transgenes. Nuclear run-on transcription analysis revealed that silencing of gus, OSM and hph genes in OSM25 operates at the transcriptional level. P35S showed hypermethylation in OSM25 plants. COT-OSM4 has P35S-driven gus and hph genes in its T-DNA. The hph gene, which is proximal to the RB, is in a divergent orientation of transcription in the IR. Unlike in OSM25, the transgenes in COT-OSM4 showed no silencing. These findings show that convergent orientation of transcription of a gene at the origin of an IR is important for transgene silencing.