Ac/Ds-transposon activation tagging in poplar: a powerful tool for gene discovery

Abstract

BackgroundRapid improvements in the development of new sequencing technologies have led to the availability of genome sequences of more than 300 organisms today. Thanks to bioinformatic analyses, prediction of gene models and protein-coding transcripts has become feasible. Various reverse and forward genetics strategies have been followed to determine the functions of these gene models and regulatory sequences. Using T-DNA or transposons as tags, significant progress has been made by using "Knock-in" approaches ("gain-of-function" or "activation tagging") in different plant species but not in perennial plants species, e.g. long-lived trees. Here, large scale gene tagging resources are still lacking.ResultsWe describe the first application of an inducible transposon-based activation tagging system for a perennial plant species, as example a poplar hybrid (P. tremula L. × P. tremuloides Michx.). Four activation-tagged populations comprising a total of 12,083 individuals derived from 23 independent "Activation Tagging Ds" (ATDs) transgenic lines were produced and phenotyped. To date, 29 putative variants have been isolated and new ATDs genomic positions were successfully determined for 24 of those. Sequences obtained were blasted against the publicly available genome sequence of P. trichocarpa v2.0 (Phytozome v7.0; http://www.phytozome.net/poplar) revealing possible transcripts for 17 variants.In a second approach, 300 randomly selected individuals without any obvious phenotypic alterations were screened for ATDs excision. For one third of those transposition of ATDs was confirmed and in about 5% of these cases genes were tagged.ConclusionsThe novel strategy of first genotyping and then phenotyping a tagging population as proposed here is, in particular, applicable for long-lived, difficult to transform plant species. We could demonstrate the power of the ATDs transposon approach and the simplicity to induce ATDs transposition in vitro. Since a transposon is able to pass chromosomal boundaries, only very few primary transposon-carrying transgenic lines are required for the establishment of large transposon tagging populations. In contrast to T-DNA-based activation tagging, which is plagued by a lack of transformation efficiency and its time consuming nature, this for the first time, makes it feasible one day to tag (similarly to Arabidopsis) every gene within a perennial plant genome.

An ActiveAc/DsTransposon System for Activation Tagging in Tomato Cultivar M82 Using Clonal Propagation

“…Mutant populations have been produced using the maize Activator (Ac)/Dissociator (Ds) system in Arabidopsis (Arabidopsis thaliana; Kuromori et al, 2004), rice (Oryza sativa; Qu et al, 2008), and poplar (Populus spp. ; Fladung and Polak, 2012) and the Enhancer-Inhibitor/ Supressor-mutator system for forward and reverse genetics in Arabidopsis (Speulman et al, 1999(Speulman et al, , 2000 and rice (Kumar et al, 2005;Krishnan et al, 2009). Use of the Ac/Ds system enables the recovery of many independent mutants from a single transformed line and facilitates the rapid identification of putatively mutagenized sequences through the isolation of Ds insertion flanking regions (Kuromori et al, 2004).…”

Genome Editing of Plants

“…The modified Suzuki et al (2001) construct (transposase-deficient) was co-transformed with a plasmid containing transposase controlled by the Glycine max L. (soybean) heat shock promoter to control transposition. In this system, a gain-of-function mutation frequency of 1% was achieved (Fladung and Polak, 2012).…”

“…Both genes were demonstrated to be active before transposition, and inactive after (Suzuki et al, 2001). Fladung and Polak (2012) modified the Suzuki et al (2001) system by incorporating an inducible transposase gene to generate a mutant population in the perennial aspen-Populus hybrid (P. tremula L. £ P. tremuloides Michx.). The modified Suzuki et al (2001) construct (transposase-deficient) was co-transformed with a plasmid containing transposase controlled by the Glycine max L. (soybean) heat shock promoter to control transposition.…”

Development ofmPing-based Activation Tags for Crop Insertional Mutagenesis

“…Our goal is to develop a transposon-based activation tagging system that overcomes these limitations. Activation tags designed around the Activator (Ac)/Dissociation (Ds) (Wilson et al, 1996;Schaffer et al, 1998;Fridborg et al, 1999;Suzuki et al, 2001;Fladung and Polak, 2012) and Enhancer/Suppressor (Marsch-Martínez, 2011) transposon systems engineered with constitutive promotors or enhancer sequences clearly demonstrate that modified transposons retain their mobility and also induce gene expression. These approaches have been used to clone a variety of genes including TINY (Wilson et al, 1996), LATE ELONGATED HYPOCOTYL (Schaffer et al, 1998), SHORT INTERNODES (Fridborg et al, 1999), high phenolic compound1-1 Dominant (Schneider et al, 2005), and a strictosidine synthase (Mathieu et al, 2009).…”