A versatile method to prevent transcriptional gene silencing in Arabidopsis thaliana

Transcriptional gene silencing (TGS) is a phenomenon by which transgenes that share homology in their promoter regions are inactivated. TGS is known to be reduced by using different promoters to drive the expression of each transgene. However, in order to perform a large-scale genetic screen in which overexpression lines are mutagenized by TDNA tagging and therefore harbor two types of transgene, it is critical to develop a technique that consistently blocks TGS. Here, I report a versatile method that completely prevents TGS in transgenic Arabidopsis. A seedling morphology-based assay demonstrated that TGS could be significantly diminished by using different terminator sequences for each transgene. Furthermore, it was suggested that TGS might be reduced if the orientations of the two T-DNA sequences were reversed relative to each other. By combining these strategies, I showed that TGS was completely blocked in over 50,000 T1 plants. These findings present a thorough and versatile method to prevent TGS that is potentially applicable to various plant species and is expected to be used in diverse situations, from basic to applied research fields.