A synthetic biology approach allows inducible retrotransposition in whole plants

Abstract

Retrotransposons are mobile genetic elements that transpose by reverse transcription of element RNA, followed by insertion of the cDNA into new positions of the host genome. Although they are major constituents of eukaryotic genomes, many facets of their biology remain to be understood. Transposition is generally rare, suggesting that it is subject to tight regulation. However, only the first regulatory step (transcriptional induction) is currently amenable to investigation in higher eukaryotes. To investigate the complete life cycle of a long terminal repeat (LTR) retrotransposon in plants, we established a synthetic biology program on tobacco retrotransposon Tto1, and achieved transposition in whole plants triggered by an inducible promoter. The engineered element, iTto (inducible Tto1), is a novel tool for analysis of retrotransposition in plants. In addition, it allows to explore the potential of an inducible retrotransposon for insertional mutagenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11693-010-9053-4) contains supplementary material, which is available to authorized users.

Exogenous Transposable Elements Circumvent Identity-Based Silencing, Permitting the Dissection of Expression-Dependent Silencing

“…When a full-length copy of this TE was transferred into the Arabidopsis genome, the element was active and fully capable of transposition (Hirochika et al, 2000;Böhmdorfer et al, 2010). However, this transposition was only observed after selection in tissue culture (where TE silencing is compromised; Tanurdzic et al, 2008), in a chromatin mutant, or when an inducible promoter is used to force TE expression, demonstrating that there is repression or silencing of this TE in wild-type Arabidopsis.…”

“…We stably transformed wild-type Col with an inducible full-length form of Tto (Böhmdorfer et al, 2010), which is targeted for silencing (iTto; see below; Figures 3 and 4). We then retransformed these iTto lines with the solo Tto LTR Dpro transgene from Figure 2A and found that this newly introduced TE is now targeted for identity-based (expression-independent) RdDM ( Figure 2E).…”

“…The "full Tto" transgene includes both complete LTRs and the entire Tto1 internal coding sequence (CDS) ( Figure 4A), as in the native tobacco genome. Two short introns were added in the CDS as tools to measure RNA processing and retrotransposon-induced cDNA formation (Böhmdorfer et al, 2010). Similar to the solo Tto LTR transgene, which shares the same LTR promoter, the full Tto transgene is expressed when transformed into wild-type Col ( Figure 4B).…”

“…In contrast to the solo Tto LTR, which produces both 21-to 22-nucleotide and 24-nucleotide siRNAs ( Figures 3B and 4C), the full Tto element produces low levels of only 24-nucleotide siRNAs in the inflorescence tissue of T1 plants ( Figure 4C; Supplemental Figure 5). A second transgene, iTto, was previously constructed using a full Tto1 element with added CDS introns and a truncated 59 LTR (Böhmdorfer et al, 2010). This element is not able to produce its own coding transcripts, but rather is under the control of an inducible promoter and has a transcriptional terminator ( Figure 4A).…”

“…The iTto and iRAN transgenes have been previously described (Böhmdorfer et al, 2010;Nuthikattu et al, 2013). For other transgenes, TE sequences were amplified from the Arabidopsis thaliana genome (iAthila LTR, Athila LTR, At1g08200 pro, and TAS3A internal), the iTto plasmid (solo Tto LTR), or the pHL414 (Levin et al, 1993) plasmid (iTf1) using Phusion High-Fidelity DNA Polymerase (NEB) and primers from Supplemental Table 2.…”