An active ac/ds transposon system for activation tagging in tomato cultivar m82 using clonal propagation

Abstract

Tomato (Solanum lycopersicum) is a model organism for Solanaceae in both molecular and agronomic research. This project utilized Agrobacterium tumefaciens transformation and the transposon-tagging construct Activator (Ac)/Dissociator (Ds)-ATag-Bar_gosGFP to produce activation-tagged and knockout mutants in the processing tomato cultivar M82. The construct carried hygromycin resistance (hyg), green fluorescent protein (GFP), and the transposase (TPase) of maize (Zea mays) Activator major transcript X054214.1 on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds-ATag element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 11,000 selfed and cv M82 backcrossed progeny was produced from the functional T0 line. This population was screened using glufosinate herbicide, hygromycin leaf painting, and multiplex polymerase chain reaction (PCR). Insertion sites of transposed Ds-ATag elements were identified through thermal asymmetric interlaced PCR, and resulting product sequences were aligned to the recently published tomato genome. A population of 509 independent, Ds-only transposant lines spanning all 12 tomato chromosomes has been developed. Insertion site analysis demonstrated that more than 80% of these lines harbored Ds insertions conducive to activation tagging. The capacity of the Ds-ATag element to alter transcription was verified by quantitative real-time reverse transcription-PCR in two mutant lines. The transposon-tagged lines have been immortalized in seed stocks and can be accessed through an online database, providing a unique resource for tomato breeding and analysis of gene function in the background of a commercial tomato cultivar.

Figure 1.

The Ac/Ds-ATag-Bar_gosGFP construct. Elements are as follows: LB, left border; Hyg, hygromycin resistance; 35SPr, cauliflower mosaic virus 35S promoter; GosPr, maize Gos2 promoter; AcTPase, maize transposase derived from GenBank accession X05424.1; AcPr, maize transposase native promoter; IR, inverted repeat; 35S 4 Enh, four tandem copies of cauliflower mosaic virus 35S enhancer; BAR, glufosinate resistance; UbiPr, ubiquitin promoter; RB, right border.

Figure 2.

Phenotypic screening of T1 progeny. A, Herbicide-resistant 6-week-old tomato seedling (left) next to a sensitive seedling (right) 7 d after spraying with 0.05% (v/v) Liberty (glufosinate) solution. B, Closeup of an herbicide-resistant leaf (left) next to a sensitive leaf (right) removed from the seedlings in A. C, Adaxial side of four leaves detached from different seedlings sensitive to hygromycin 6 d after painting with 100 mg L⁻¹ hygromycin + two drops of polysorbate 20. Reaction to hygromycin was localized to the region of a leaf that had been painted. D, Abaxial side of a leaf sensitive to hygromycin displaying the diagnostic necrotic spots that distinguished hygromycin sensitivity from common leaf damage due to pathogens or mechanical injury.

Figure 3.

Analysis of transgenic and transposon-tagged lines by RT-PCR, multiplex PCR, and TAIL-PCR. A, Expression of transposase in whole inflorescence RNA. The PCR product spans transposase intron 2 (DNA product = 476 bp, cDNA product = 405 bp). Positive control DNA and cDNA extracted from transgenic potato inflorescence are shown. B, Screening of 13 T1 progeny (lanes 3–14) by multiplex PCR and zygosity testing. SQE, Squalene epoxidase (distinguishes the wild types from failed reactions); CI#1, common insertion 1 from a somatic transposition event early in development (defines T0 group 2); BAR, glufosinate herbicide resistance gene (Ds element); HPT, hygromycin resistance gene (Ac element); CI#2, common insertion 2 from somatic transposition event unique to T0 7m. Plants in lanes 13 and 14 represent potentially unique transposants destined for TAIL-PCR. Lanes 1 and 16 are the 1-kb ladder; lane 2 is master mix without DNA (negative control). C, Visualization of primary TAIL-PCR products. Lanes 2 to 16 (T1 7g-98–T1 7b-84) are amplification products obtained from 16 independently identified Ds-only, BAR-positive, hyg-sensitive T1 plants; lanes 1 and 17 are the 2-kb ladder.

Figure 4.

Graphic distribution of 509 transposed Ds elements to tomato pseudochromosomes (Tomato WGS Chromosomes SL2.40). Self and wild type × T0 progeny from every T0 family are included. Both common and unique insertions are represented by a single band. The original T-DNA insertion site during transformation (Ac element) is denoted by ←Ac on chromosome 5. Locations of common somatic insertions are denoted by triangles.

Figure 5.

Insertion sites of the Ds element for mutant lines Wt7i-15 and 7i-203 (Tomato WGS Chromosomes SL2.40). −, Both Ds element inserted. Primer sites for quantitative real-time RT-PCR are denoted by arrows below the exons.

Figure 6.

Phenotypes for seven T1 activation-tagged mutants. A, 7c-79: rolled leaves, no lateral branching, weak stem. B, Wt7i-15: twisted, soft leaves. C, 7g-300: crumpled leaves. D, 7i-271: twisted leaves, extreme dwarfism. E, 7g-25: tightly rolled, leathery leaves, dwarf, aborted flowers. F, Wt7i-48: extensive yellow variegation, aborted flowers. G, 7i-413: leaves, internodes, inflorescences, fruit, and seeds proportionally reduced in size.