Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing

Vojtěch Čermák,Lukáš Fischer

doi:10.1186/s12870-018-1482-3

Abstract

BackgroundPlant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines.ResultsHere we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana.ConclusionsOur results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.

Highlights

Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome
The observation of pervasive read-through transcription of T-DNAs that we describe in this study, was discovered during our RNA interference (RNAi) project focused on comparison of silencing potential of different silencing inducers
Fluorescence in calli transformed with various GFP silencer constructs To study various aspects of RNAi, we prepared three different silencer T-DNAs based on the XVE inducible system [26]

Summary

Introduction

Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. Agrobacterium tumefaciens mediated transformation is a common method used to obtain transgenic plants. Agrobacterium transfers its T-DNA into a plant cell, where it can be integrated inside plant genome predominantly through double-strand break repair pathway (reviewed in [1]). The T-DNAs are integrated at random positions in their genome [2, 3]. Due to the way how T-DNAs are integrated, the most common form is the head-to-head (RB-to-RB) inverted repeat arrangement [9, 10]. Convergent read-through transcription of T-DNAs integrated as inverted repeats can induce silencing of homologous sequences via RNA interference (RNAi) [11, 12]

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Results

Discussion

Conclusion