Transgene structures suggest that multiple mechanisms
are involved in T-DNA integration in plants
Qian-Hao Zhu
a,b
, Kerrie Ramm
a,b
, Andrew L. Eamens
a,1
,
Elizabeth S. Dennis
a,b
, Narayana M. Upadhyaya
a,b,
*
a
CSIRO Plant Industry, Canberra, ACT 2601, Australia
b
New South Wales Agricultural Genomics Centre, Wagga Wagga, NSW 2678, Australia
Received 19 September 2005; received in revised form 20 January 2006; accepted 28 March 2006
Available online 24 April 2006
Abstract
To gain further understanding of the mechanisms involved in Agrobacterium-mediated genetic transformation and T-DNA integration, we
analysed 156 T-DNA/rice, 69 T-DNA/T-DNA and 11 T-DNA/vector backbone (VB) junctions, which included 171 left borders (LB) and 134 right
borders (RB). Conserved cleavage was observed in 6% of the LB and 43% of the RB. Terminal microhomology (1–10 bp) was identified in 58% of
T-DNA/rice, 43% of T-DNA/T-DNA and 82% of T-DNA/VB junctions, and this occurred particularly at the LB junctions. Approximately 32% of
both T-DNA/rice and T-DNA/T-DNA junctions harboured 1–344 bp of filler DNA that was derived mainly from the T-DNA region adjacent to the
breakpoint and/or from the rice genome flanking the T-DNA integration site. Structure of the filler DNA was more complicated at the T-DNA/T-
DNA junction than at the T-DNA/rice junction, indicating the presence of T-DNA recombination or rearrangement prior to or during T-DNA
integration. When two T-DNAs were integrated in the inverted repeat configuration, significant truncation was always observed in one of the two T-
DNAs whereas with direct repeat configuration, a large truncation was less frequent. Most integration events analysed in this study could be
addressed by previously proposed models; however, the characteristics of the T-DNA repeats and the complicated filler DNA between two T-DNA
copies imply that multiple mechanisms are involved in the formation of T-DNA repeats as well as in T-DNA integration in plants.
# 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Filler DNA; Rice; T-DNA integration; T-DNA junction; Terminal microhomology
1. Introduction
Agrobacterium tumefaciens is well known for its capacity
for inter-kingdom DNA transfer [1]. It harbours a tumour-
inducing (Ti) plasmid that encodes most of the major functions
required for transferring an oncogenic segment of DNA, the
transferred DNA (T-DNA), into the host cell [2,3]. The T-DNA
itself does not include genes required for this transfer process
and is delimited by two 25-bp imperfect terminal repeats,
termed T-DNA left and right borders (LB and RB). The wild-
type T-DNA sequence between the borders has been deleted
and specifically modified to provide a range of vectors for
introducing genes of interest into dicotyledonous and mono-
cotyledonous plants, and as a genetic tool for functional
genomics in plants. In view of the importance of Agrobacter-
ium-mediated plant genetic engineering, a great deal of effort
has been devoted to unravelling the mechanism of T-DNA
integration.
T-DNA integration initiates with the induction of Vir genes
in Agrobacterium. A single-stranded (ss) T-strand is released
from the Ti plasmid by the border-specific action of the VirD1/
VirD2 protein complex, and the VirD2 moiety covalently
attaches to the 5
0
end of the T-strand. The T-strand is then
delivered across the bacterial envelope and into the plant cell
using a type IV secretion system (T4SS). Recently, two classes
of plant proteins (BTI and AtRAB8) that may participate in the
www.elsevier.com/locate/plantsci
Plant Science 171 (2006) 308–322
Abbreviations: ds, double-stranded; DSB, double-strand break; DSBR,
double-strand break repair; LB, T-DNA left border; NHEJ, non-homologous
end-joining; RB, T-DNA right border; ss, single-stranded; SSA, single-strand
annealing; SDSA, synthesis-dependent strand annealing; SSGR, single-strand
gap repair; T-DNA, transferred DNA; T-strand, transferred DNA strand; VB,
vector backbone; Vir, virulence
* Corresponding author. Tel.: +61 2 6246 5491; fax: +61 2 6246 5000.
E-mail address: Narayana.upadhyaya@csiro.au (N.M. Upadhyaya).
1
Present address: Department of Biology, University of York, Heslington,
York YO10 5DD, UK.
0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2006.03.019