Biomedical Engineering Reference
In-Depth Information
T-DNA
T-DNA
Nopaline
catabolism
Virulence
genes
Conjugative
transfer
Virulence
genes
Octopine
Ti-plasmid
pTiACH5
Nopaline
Ti-plasmid
pTiC58
Octopine
catabolism
Agropine
catabolism
Agrocinopine
catabolism
Conjugative transfer
Fig. 12.8
Ti-plasmid gene maps.
about 100 nucleotides (Yadav
et al.
1982, Zambryski
et al.
1982). Deletion of the right border repeat abol-
ishes T-DNA transfer, but the left-hand border sur-
prisingly appears to be non-essential. Experiments
in which the right border repeat alone has been used
have shown that an enhancer, sometimes called the
overdrive sequence
, located external to the repeat is
also required for high-efficiency transfer (Shaw
et al.
1984, Peralta
et al.
1986). The left border repeat has
little transfer activity alone ( Jen & Chilton 1986).
O
CH
3
O
CH
2
OH
C
C
CH
3
O
OCH
3
CH
3
O
OCH
3
OH
OH
Acetosyringone
α
-hydroxyacetosyringone
Fig. 12.9
Structures of signal molecules, produced by
wounded plant tissue, which activate T-DNA transfer by
Agrobacterium tumefaciens
.
Genes required (
in trans
) for transfer
The genes responsible for T-DNA transfer are located
in a separate part of the Ti plasmid called the
vir
(virulence) region. Two of these genes,
virA
and
virG
, are constitutively expressed at a low level and
control the plant-induced activation of the other
vir
genes. VirA is a kinase that spans the inner bacterial
membrane and acts as the receptor for certain
phenolic molecules that are released by wounded
plant cells. A large number of such compounds have
been characterized, but one in particular, acetosy-
ringone, has been the most widely used in the labor-
atory to induce
vir
gene expression (Stachel
et al.
1985; Fig. 12.9). Notably, phenolic compounds such
as acetosyringone do not attract bacteria to wounded
plant cells. Rather, the bacteria appear to respond to
simple molecules, such as sugars and amino acids,
and the
vir
genes are induced after attachment
(Parke
et al.
1987, Loake
et al.
1988). Many sugars
also synergize the action of the phenolic signals to
enhance
vir
gene expression (Shimada
et al.
1990).
Activated VirA transphosphorylates the VirG pro-
tein, which is a transcriptional activator of the other
vir
genes. The VirA and VirG proteins show similar-
ities to other two-component regulatory systems
common in bacteria (Winans 1992). In addition to
virG
, further genes on the bacterial chromosome
also encode transcription factors that regulate
vir
gene expression (reviewed by Kado 1998).
The induction of
vir
gene expression results in the
synthesis of proteins that form a conjugative pilus
through which the T-DNA is transferred to the plant
cell. The components of the pilus are encoded by
genes in the
virB
operon (reviewed by Lai & Kado
2000). DNA transfer itself is initiated by an endonu-
clease formed by the products of the
virD1
and
virD2
genes. This introduces either single-strand nicks or
a double-strand break at the 25 bp borders of the
T-DNA, a process enhanced by the VirC12 and VirC2
proteins, which recognize and bind to the overdrive
enhancer element. The VirD2 protein remains cova-
lently attached to the processed T-DNA. Recent studies
have suggested that the type of T-DNA intermediate
produced (single- or double-stranded) depends on
the type of Ti plasmid, with double-stranded T-DNA
favoured by nopaline plasmids (where the T-DNA is
a single element) and single 'T strands' favoured by
