Structural features of T-DNA that induce transcriptional gene silencing during agroinfiltration

Agrobacterium tumefaciens (Rhizobium radiobacter) is used for the transient expression of foreign genes by the agroinfiltration method, but the introduction of foreign genes often induces transcriptional and/or post-transcriptional gene silencing (TGS and/or PTGS). In this study, we characterized the structural features of T-DNA that induce TGS during agroinfiltration. When A. tumefacienscells harboring an empty T-DNA plasmid containing the cauliflower mosaic virus (CaMV) 35S promoter were infiltrated into the leaves of Nicotiana benthamiana line 16c with a GFP gene over-expressed under the control of the same promoter, no small interfering RNAs (siRNAs) were derived from the GFP sequence. However, siRNAs derived from the CaMV 35S promoter were detected, indicating that TGS against the GFP gene was induced. When the GFP gene was inserted into the T-DNA plasmid, PTGS against the GFP gene was induced whereas TGS against the CaMV 35S promoter was suppressed. In other words, depending on the combination of promoter and coding sequences on T-DNA and the host nuclear genome, either or both TGS and/or PTGS could be induced by agroinfiltration. We also showed the importance of terminator sequences in T-DNA for gene silencing and the possible involvement of three siRNA-producing Dicers in the induction of TGS. These results are valuable for controlling gene expression by agroinfiltration.