Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration

Abstract

Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T-DNA), derived from its tumour-inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T-DNA also to monocotyledonous plants, yeasts and fungi. The T-DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T-DNA recipient to demonstrate that the non-homologous end-joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome. We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements.

Fig. 1. Schematic representation of the T-DNA from pSDM8000. The T-DNA from pSDM8000 was used in co-cultivation experiments to study T-DNA integration by NHR in recombination defective S.cerevisiae strains. The T-DNA contains the KanMX cassette, which consists of the kan resistance gene of the Escherichia coli transposon Tn903 under control of transcriptional and translational sequences of the filamentous fungus Ashbya gossypii TEF gene. This module allows selection of S.cerevisiae transformants resistant against the antibiotic G418 (Wach et al., 1994).

Fig. 2. T-DNA integrates preferentially at (sub)telomeric regions in rad50, mre11 and xrs2 mutants. (A) Schematic representation of a yeast chromosome end, showing the position of the telomeric (G_1–3T) repeats, the Y′ and X elements and the centromere. Y′ and X elements are present at only a subset of all chromosomes and are often found in association with internal tracts of (G_1–3T) repeats. (B) Junction sequences of T-DNA left end and genomic DNA of the S.cerevisiae rad50, mre11 and xrs2 mutants. Genomic DNA sequences are shown in italics, T-DNA sequences in normal capitals. Bold sequences represent microhomology of the T-DNA left end with the integration site. Filler DNA sequences are underlined and depicted in italics. The numbers above the sequences represent the number of base pairs deleted from the T-DNA left end. Tel. = telomeric region; Subtel. = subtelomeric region; rDNA = ribosomal DNA region; LTR = long terminal repeat of Ty element; – = none of the 16 chromosomes showed an altered mobility; n.d. = not determined; LB′ = remnant of T-DNA left border repeat.

Fig. 3. T-DNA forms repeat structures in the genome of a lig4 mutant. Sequences of T-DNA structures as found in the genome of the S.cerevisiae lig4 lines are depicted. Sequences of the binary vector are underlined and depicted in italics. T-DNA sequences are shown in italics, except for the border sequences. The right border repeat (RB) is presented in bold for lig4k.2 and lig4k.3 to distinguish between RB and LB sequences. RB′ and LB′ = fused remnants of the RB and LB, respectively.

Fig. 4. The position of T-DNA integration by NHR in the genome of a sir4 mutant is not biased. Junction sequences of the T-DNA left end and genomic DNA of the sir4 mutant are depicted. Genomic DNA sequences are shown in italics, T-DNA sequences in normal capitals. The numbers above the sequences represent the number of base pairs deleted from the left T-DNA end.

Fig. 5. T-DNA integration is accompanied by chromosomal rearrangements in rad50, mre11, xrs2 and lig4 mutants. CHEF-gel analysis of T-DNA integration events in the genome of S.cerevisiae rad50, mre11, xrs2, lig4 and sir4 mutants was studied. Chromosomes from G418-resistant S.cerevisiae colonies obtained after co-cultivation with A.tumefaciens carrying pSDM8000 were isolated, separated on a CHEF gel (left panels in A and B) and blotted on a membrane. The membrane was hybridized with a labelled KanMX probe that anneals to the T-DNA and an autoradiograph was made (right panels in A and B). (A) T-DNA transformed rad50 and lig4 mutants and their isogenic and untransformed wild-type control (C). (B) T-DNA transformed mre11, xrs2 and sir4 mutants and their isogenic and untransformed wild-type control (C). White arrowheads indicate chromosomes with an altered mobility.