A PTGS locus of an inverted T-DNA repeat in the transgenic rice line CG57 causes PTGS-triggered trans-TGS of transgenes which are driven by homologous promoters

We report a complex pattern of silenced and non-silenced transgenes in a transgenic rice plant CG57 that harboured a right border (RB)-centered inverted T-DNA repeat (IR). The T-DNA of CG57 has four transgenes. In the RB-centered IR (RB-IR), the RB-proximal P35S- gus - nos 3’ genes form a transcriptionally convergent dimer at the origin of IR (OIR). The P35S- gus dimer is flanked on either side by three transgenes, P Ubi 1- chi 11- chi 113’, P35S- GLU -35S3’, and P35S- hph -35S3’. CG57 displays an unusual mosaic pattern of silencing and methylation. The P35S-transgenes (P35S- gus , P35S- GLU , and P35S- hph ) are silenced, whereas the P Ubi 1- chi 11 gene is not silenced. Interestingly, silencing and methylation in P35S-transgenes are not uniform despite high sequence homology in their P35S promoters. Run-on transcription analysis revealed that the P35S- gus is under PTGS, while the P35S- GLU and P35S- hph are under TGS. The P35S- gus gene under PTGS displays coding region methylation, while the P35S- GLU and P35S- hph genes under TGS display methylation in the P35S promoter region. Additionally, CG57 accumulated siRNA against the PTGS P35S- gus gene but not against the TGS P35S- hph gene. We propose that the PTGS silenced P35S- gus gene triggered TGS over other homologous 35S promoter-driven GLU and hph transgenes. CG57 (silencing trigger) was crossed with two other single-copy transgenic rice lines, TC7 and TC17 (silencing targets) which harboured the active transgenes, P35S- cht 42-35S3’ and P35S- hph -35S3’. In F ₁ hybrids, barring P35S- gus, all P35S-driven transgenes displayed PTGS-triggerred trans silencing. The target loci ( TC7 / TC17 ) with silenced P35S- cht 42 and P35S- hph transgenes in the F ₁ plants did not inherit silencing in the F ₂ segregants in which the trigger locus ( CG57 ) was segregated out. Evaluation of silencing in CG57 shows that P35S of the PTGS-silenced P35S- gus gene triggers TGS of other linked and unlinked genes which are transcribed by P35S.