Formation of complex extrachromosomal T-DNA structures in Agrobacterium tumefaciens-infected plants

Abstract

Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciens-mediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown "filler" sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant's DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate.

Figure 1.

Constructs and experimental procedure for the isolation of T-circles. A, Schematic diagram of the three T-DNA constructs used throughout the experiments: AMP-ORI, Amp^R with ori; KAN-ORI, Kan^R with ori; KAN, Kan^R without ori. B, Illustration of the experimental procedure: 1, transformation of A. tumefaciens with a pRCS2 binary plasmid harboring one of the three T-DNA constructs (AMP-ORI is shown); 2, agroinfiltration of plant leaves; 3, extraction of DNA from the agroinfiltrated leaves (4–8 d after infiltration); 4, transformation of E. coli with the DNA; 5, selection of colonies resistant to ampicillin (or kanamycin if KAN-ORI or KAN is used); 6, detection of spectinomycin/streptomycin-sensitive colonies; 7, isolation of plasmids from each colony and analysis by restriction digestion and DNA sequencing. amp, Ampicillin; kan, kanamycin; spec, spectinomycin; strep, streptomycin. [See online article for color version of this figure.]

Figure 2.

Restriction digestion reveals T-circles of various sizes and structures. A, Agroinfiltration of tobacco with AMP-ORI (T-1–T-6). B, Coagroinfiltration of N. benthamiana with AMP-ORI and KAN (T-7, T-8, T-10, and T-11) or AMP-ORI and KAN-ORI (T-9 and T-12–T-16). T-circles are shown on agarose gels as uncut (U; left lane), ScaI treated (S; middle lane), or ClaI treated (C; right lane). Each T-circle is marked by + or − (below the gel) to indicate the resistance or sensitivity (respectively) to an antibiotic (left side) it confers to E. coli. The binary plasmids AMP-ORI and KAN-ORI are shown (last two on bottom right). C, Schematic diagram of T-7 shows a junction fragment of AMP-ORI and KAN. Numbers in parentheses represent the positions within the T-DNA relative to the LB (0 point) to indicate the deleted regions from each side. [See online article for color version of this figure.]

Figure 3.

A. tumefaciens T-DNA transfer is required for T-circle formation. A, A T-DNA construct with the GUS sequence interrupted by an intron was introduced into A. tumefaciens strains virB9^–, virB9^–/pED37, and EHA105 (the control strain). GUS staining was performed 3 d after agroinfiltration. B, The AMP-ORI construct was introduced into each of the three A. tumefaciens strains, which were then agroinfiltrated into N. benthamiana. Ampicillin-resistant E. coli colonies were selected and tested for spectinomycin/streptomycin resistance. The numbers in the pie charts are given as percentages of colonies. S, Spectinomycin/streptomycin; +, resistant; −, sensitive. [See online article for color version of this figure.]

Figure 4.

T-circles containing both AMP-ORI and KAN-ORI constructs form in plants following coagroinfiltration. A, Schematic illustration of the locations of the primers designed for the AMP-ORI and KAN-ORI constructs. The primers P1a, P1k, P2a, and P2k are positioned near the T-DNA ends (arrows indicate the directions of the primers). B, DNA samples were extracted from N. benthamiana agroinfiltrated with AMP-ORI (I), agroinfiltrated with KAN-ORI (II), or coagroinfiltrated with both AMP-ORI and KAN-ORI (co-agro). The fourth DNA sample is a mixture of DNA from I and II (mix). C, PCR amplification using as a template DNA from the samples I, II, co-agro, mix, and (as a control) double distilled water (ddw). The primers used in each reaction are indicated on the right side of each gel. [See online article for color version of this figure.]

Figure 5.

Antibiotic resistance of E. coli colonies transformed with DNA I, II, co-agro, and mix (the DNA sample types are indicated above the pie charts). Colonies were initially selected on either ampicillin or kanamycin (indicated to the left of the pie charts) and then tested for spectinomycin/streptomycin, ampicillin, and kanamycin resistance. The numbers in the pie charts are given as percentages of colonies. The numbers of colonies tested in each group are follows: a, n = 96; b, n = 478; c, n = 383; d, n = 120; e, n = 473; f, n = 477. A, Ampicillin; K, kanamycin; NC, no colonies; S, spectinomycin/streptomycin; +, resistant, −, sensitive. [See online article for color version of this figure.]

Figure 6.

Schematic illustration of the T-circles T-1 to T-6 based on DNA sequencing. A, T-1 to T-5 are shown in a linear form, opened at the position of a junction, and aligned below a complete AMP-ORI construct. B, T-6 after digestion with BsaXI (left lane) or AatII (right lane). The bands that include the junctions are indicated by arrows. C, Schematic illustration of the AatII fragment (top; the LB-LB region junction of T-6) and the BsaXI fragment (bottom; the RB-RB region junction of T-6). [See online article for color version of this figure.]

Figure 7.

A proposed model for T-DNA integration, the formation of complex T-DNA structures, and T-circles. 1, In A. tumefaciens, the T-DNA borders are nicked by the VirD1-VirD2 endonuclease complex and an ss T-DNA (T-strand) is excised (the T-DNAs are marked in red). A VirD2 protein remains attached to the 5′ terminus of the T-strand (the RB). 2, The T-strand is transferred into the plant cell nucleus. Additional ss DNA molecules (derived from the A. tumefaciens binary plasmid or chromosomal DNA) may occasionally be transferred independently or together with a T-DNA by read-though linkage. 3, In the nucleus, ss DNAs are converted into ds DNAs (only ds T-DNAs are shown). 4, ds DNA may then integrate into the genome (first outcome) via VirD2 and the plant’s DSB-repair pathway. 5, Termini of a ds DNA may ligate to each other (second outcome) and generate T-circles. 6, Termini of different ds DNA molecules may ligate to each other (third outcome) and form complex structures. These structures may then integrate or form complex circular molecules. [See online article for color version of this figure.]