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

## TL;DR

This study explores how the structure of T-DNA in agroinfiltration affects gene silencing, revealing how promoter, terminator, and gene sequences influence transcriptional and post-transcriptional gene silencing.

## Contribution

The study identifies structural features of T-DNA that trigger transcriptional gene silencing during agroinfiltration.

## Key findings

- T-DNA with a CaMV 35S promoter induced TGS but not PTGS when the GFP gene was absent.
- Inserting the GFP gene into T-DNA induced PTGS and suppressed TGS against the promoter.
- Terminator sequences in T-DNA play a key role in gene silencing, and DCL2 is likely crucial for TGS induction.

## Abstract

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. tumefaciens cells 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. We also showed the importance of terminator sequences in T-DNA for gene silencing. Therefore, depending on the combination of promoter, terminator and coding sequences on T-DNA and the host nuclear genome, either or both TGS and/or PTGS could be induced by agroinfiltration. Furthermore, we showed the possible involvement of three siRNA-producing Dicers (DCL2, DCL3 and DCL4) in the induction of TGS by the co-agroinfiltration method. Especially, DCL2 was probably the most important among them in the initial step of TGS induction. These results are valuable for controlling gene expression by agroinfiltration.