Efficient repair of genomic double-strand breaks by homologous recombination between directly repeated sequences in the plant genome

Abstract

Previous studies demonstrated that in somatic plant cells, homologous recombination (HR) is several orders of magnitude less efficient than nonhomologous end joining and that HR is little used for genomic double-strand break (DSB) repair. Here, we provide evidence that if genomic DSBs are induced in close proximity to homologous repeats, they can be repaired in up to one-third of cases by HR in transgenic tobacco. Our findings are relevant for the evolution of plant genomes because they indicate that sequences containing direct repeats such as retroelements might be less stable in plants that harbor active mobile elements than anticipated previously. Furthermore, our experimental setup enabled us to demonstrate that transgenic sequences flanked by sites of a rare cutting restriction enzyme can be excised efficiently from the genome of a higher eukaryote by HR as well as by nonhomologous end joining. This makes DSB-induced recombination an attractive alternative to the currently applied sequence-specific recombination systems used for genome manipulations, such as marker gene excision.

Figure 1.

Scheme of the T-DNA from the Binary Plasmid pGU.C.USB. A codA gene flanked by two I-SceI sites was integrated between overlapping halves of a GUS gene. The T-DNA used for transformation also contained the bar gene as a transformation marker. Possible outcomes of the excision reaction of the codA marker gene are depicted. Triangles represent the primers used for PCR amplification of the recombined junctions. A 0.7-kb fragment is indicative of HR, and a 1.4-kb fragment is indicative of NHEJ. Restriction sites used for DNA gel blot analysis were Acc65I (A) and HindIII (H) (see Figure 4). A 3.7-kb Acc65I GUS-specific fragment is indicative of HR, and a 4.4-kb fragment is indicative of NHEJ. LB, left border; RB, right border.

Figure 2.

GUS Assay of 5-FU–Resistant Calli. Transformants in which the marker gene was removed by HR result in blue staining of the whole callus. The small sectors visible in some of the other calli are caused by secondary recombination events that occurred during callus growth. The calli tested and the experiment numbers are as follows: GU.C.USB 3-1 (A1), 3-3 (A2), 3-5 (A3), 3-6 (A4), 3-7 (A5), and 3-18 (A6); GU.C.USB 7-14 (B1), 7-34 (B2), 7-58 (B3), 7-96 (B4), and 7-102 (B5); GU.C.USB 1-83 (C1), 1-61 (C2), 1-60 (C3), 1-55 (C4), and 1-23 (C5); and GU.C.USB 3-10 (D1), 3-18 (D2), 3-19 (D3), 3-21 (D4), and 3-65 (D5). As a positive control, callus of pBG5 (transgenic tobacco with a functional GUS gene [Puchta et al., 1995b]) was used (C6); as a negative control, callus of the nontransformed tobacco line SR1 was used (B6). In accord with the histochemical staining, the molecular analysis of lines GU.C.USB 1-83, GU.C.USB 3-3, 3-7, and 3-18, and GU.C.USB 7-34 by PCR and DNA gel blot analysis revealed the restoration of the GUS gene by HR (see also Figure 3). PCR and DNA gel blot analysis of lines GU.C.USB 1-23, 1-55, and 1-61, GU.C.USB 3-1, 3-5, 3-6, 3-10, 3-19, 3-21, and 3-65, and GU.C.USB 7-14 and 7-102 demonstrated that the elimination of the codA gene occurred by NHEJ (see also Figure 3). PCR analysis of lines GU.C.USB 1-60 and GU.C.USB 7-58 and 7-96 resulted in the 1.4-kb band indicative of NHEJ, whereas in the case of line GU.C.USB 3-65, the lack of a PCR product indicated a major rearrangement of the transgene locus.

Figure 3.

DNA Gel Blot Analysis with Restriction Digested DNA of Plant Lines GU.C.USB 1, 3, and 7 and Some Recombinants. Lane 1, GU.C.USB 1; lane 2, GU.C.USB 1-61; lane 3, GU.C.USB 1-83; lane 4, GU.C.USB 3; lane 5, GU.C.USB 3-1; lane 6, GU.C.USB 3-3; lane 7, GU.C.USB 7; lane 8, GU.C.USB 7-14; lane 9, GU.C.USB 7-34. The faint band at ∼6 kb in all nine lanes in (B) is attributable to a HindIII-specific fragment present in wild-type tobacco that cross-hybridizes weakly with the codA-specific probe. (A) HindIII-restricted DNA hybridized with a GUS-specific probe. (B) HindIII-restricted DNA from (A) washed to remove the GUS probe and rehybridized with a codA-specific probe. (C) Acc65I-restricted DNA hybridized with a GUS-specific probe. (D) Acc65I-restricted DNA from (C) washed to remove the GUS probe and rehybridized with a codA-specific probe.

Figure 4.

Compilation of Five Junctions Originating from GU.C.USB 1 in Which End Joining between the Two I-SceI Sites Had Occurred. Nucleotides of the I-SceI recognition sequence are highlighted in red. The 5′ 13 nucleotides of the recognition site originate from the proximal I-SceI site, and the remaining five nucleotides originate from the distal I-SceI site, of pGU.C.USB.