infected region to prevent spread of the invasion. Pathogens, in turn, have developed
methods to introduce regulatory compounds into plant cells, in an attempt to shut down
the defensive machinery of the target cell. Although some of these mechanisms are
known, some are still being investigated, and a more thorough understanding of the infec-
tion process will allow further increases in the efficiency ofAgrobacterium-mediated
transformation.
Once thevirgenes are activated, the T-DNA on the binary vector is processed for
transport to the target plant cell. Some of thevirgene products excise the T-DNA from
the binary plasmid as a single-stranded DNA molecule, while othervirgene products
coat the T-DNA to prevent degradation. Yet additionalvirgene products bind to the T-
DNA to act as navigators or signals to direct the DNA out of the bacterium, through the
plant cytoplasm, and to the nucleus. Through the action of othervirgenes, the bacterium
produces apillus, which is the conduit for transfer of the T-strand (the single-stranded,
coated, signal containing T-DNA is the “T-strand”) from the bacterium to the target
plant cell. The pillus is essentially a protein tube, which extends from the bacterium
through the cell wall and into the cytoplasm of the target cell. After the T-strand is delivered
to the nucleus, the last role of the signal protein on the T-strand is to find and nick the
host DNA as an insertion point for the T-DNA. The T-DNA appears to insert primarily
into gene-rich and transcriptionally active regions of DNA that are more exposed
and accessible.
10.3.3 Optimizing Delivery and Broadening the Range of Targets
As more is learned about the mechanisms underlyingAgrobacterium-mediated transform-
ation of plant cells, the efficiency of the process will undoubtedly increase. The three main
approaches for improving transformation are (1) increase delivery of the bacteria, (2) induce
thevirgenes, and (3) minimize defense responses of the target tissue.
Numerous methods have been developed to increase the delivery of the bacteria to the
target plant tissue. Since the bacteria infect though wounded tissues, most of these
methods strive to either increase overall wounding or call for precision wounding. The
most common tool for wounding of the target tissue is the scalpel, which is simply used
to excise plant tissues. When the tissue is cut, this presents a suitable binding/entry
point for the bacterium. Wounding can be increased by scoring the target tissue multiple
times, with a scalpel blade. Severe wounding of this sort eventually leads to a loss of the
ability of the plant tissue to regenerate. Precision wounding using either sonication or par-
ticle bombardment (described later in this chapter) results in the generation of large
numbers of extremely small wounds. Precision microwounding, if done properly, does
not extensively damage the tissue structure and tremendously increases the number of
entry points and attachment sites for the bacteria.
Induction of thevirgenes through the addition of acetosyringone has led to routine trans-
formation of plants that were initially not thought to be susceptible toAgrobacterium-
mediated transformation. Although acetosyringone may not improve transformation of
very susceptible plants (which already produce sufficient levels), it is routinely added
during the coculture period for most other plants.Cocultureis the time period during
which bacteria are permitted to invade, infect, and transform plant cells. The coculture
period ends when appropriate antibiotics are added, to eliminate the bacteria after their
job is done. Results, similar to acetosyringone addition, can be obtained with the use of
virgene mutants, which were modified to be active in the absence of acetosyringone.
10.3. AGROBACTERIUM 253