- It is circular and not always
- It’s contains 56%GC content out of which 81% encodes for a protein.
- It has various types of opines
- Octopine
- Nopaline
- Succinamopine
- Leucinopine
- It is found in Agrobacterium tumefacien and Agrobacterium rhizogenes.
- There is no pseudogene.
- It works only in dicotyledon plants.
- The Ti plasmid is lost when Agrobacterium is grown above 28°C. Such cured bacteria do not induce crown galls, i.e. they become avirulent.
Genes in the virulence region are grouped into the operons vir ABCDEFG, which code for the enzymes responsible for mediating transduction of T-DNA to plant cells.
- virA codes for a receptor which reacts to the presence of phenolic compounds such as acetosyringone, syringealdehyde oracetovanillone which leak out of damaged plant tissues.
- virB encodes proteins which produce a pore/pilus-like structure.
- virC binds the overdrive sequence.
- virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA segment, beginning with the right border.
- virG activates vir-gene expression after binding to a consensus sequence, once it has been phosphorylated by virA.
When Agrobacterium infects plants, a region of the Ti
plasmid called the T-DNA is taken up by the plant cell and incorporated into
one of its chromosomes.
The genes in the T-DNA are referred to as phyto-oncogenes because they
induce neoplastic, or tumor-producing, growth. To use the Ti plasmid as a
vector for introducing new genes into plants, it is necessary to disarm the
plasmid so that it does not cause tumors. The task is accomplished by deleting
the genes in the T-DNA that encode the enzymes controlling auxin and cytokinin
synthesis. Genes for antibiotic resistance are normally used for this purpose.
A cloned gene can then be inserted into the T-DNA of the engineered Ti
plasmid, and the plasmid can be used to infect cultured cells, leaf discs, or
root slices. The infected cells are placed on a culture medium that contains
auxin and cytokinin (to induce growth) and the antibiotic. Only the transformed
cells can grow in the presence of the antibiotic, because they have received
the T-DNA containing not only the foreign gene but also the gene for antibiotic
resistance.
To obtain a plant containing the foreign gene, it is necessary to
regenerate whole plants from the cultured, transformed cells. Fortunately,
methods for accomplishing this regeneration have been developed for many
plants, although not for all important crop species yet. The regeneration
involves adjusting the ratio of cytokinin to auxin to stimulate both shoot and
root formation.
Although the transformation of cereal crops with Agrobacterium and
their regeneration from cultured cells has been difficult, some remarkable
successes have resulted from this approach. Investigators have introduced
numerous foreign genes into plants such as tobacco, including soybean storage
protein genes. Genes for such desirable characteristics as disease resistance,
herbicide resistance, and salt tolerance have been transferred to crop plants
by these techniques, and commercial crops now are being grown with these
genetically engineered strains.
Arabidopsis can also be transformed directly by exposing whole plants to a
solution containing Agrobacterium that is carrying engineered
or wild-type Ti plasmids. The plants must be treated in such a way to allow
the Agrobacterium to enter tissue, either by applying a vacuum
or by treating with detergents. The Agrobacterium penetrates
the floral tissue and transforms the developing ovules. Isolation of seeds from
these Agrobacterium-exposed plants yields up to 2% of the seeds
that are transformed with the T-DNA. This approach is very useful for molecular
genetic studies, such as for characterizing DNA sequences involved in the
control of gene expression, or constructing large libraries of insertional
mutants.
Gene transfer using Ti plasmids
In the Ti plasmid itself, the T-DNA is flanked by 25 bp imperfect direct
repeats known as border sequences, which are conserved
between Octopine and nopaline plasmids. The border sequences are not
transferred intact to the plant genome, but they are involved in the transfer
process. The analysis of junction regions isolated from plant genomic DNA has
shown that the integrated T-DNA end-points lie internal to the border
sequences.
The right junction is rather precise, but the left junction can vary by
about 100 nucleotides. Deletion of the right border repeat abolishes T-DNA
transfer, but the left-hand border surprisingly 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.
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.
Acetosyringone, a phenolic compound has been the most widely used in the
laboratory to induce vir gene expression
But 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
Activated VirA transphosphorylates the VirG protein, which is a
transcriptional activator of the other vir genes.
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.
DNA transfer itself is initiated by an endonuclease formed by the
products of thevirD1 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 VirC1 and VirC2 proteins, which
recognize and bind to the overdrive enhancer element. The VirD2 protein remains
covalently attached to the processed T-DNA.
T strands are coated with VirE2, a single-stranded DNA-binding protein.
The whole complex, sometimes dubbed the firecracker complex because
of its proposed shape, is then transferred through the pilus and into the plant
cell. It has been proposed that the VirD2 protein protects the T-DNA against
nucleases, targets the DNA to the plant-cell nucleus and integrates it into the
plant genome. The protein has two distinct nuclear localization signals, with
the C terminal signal thought to play the major role in targeting the T-DNA.
It has been observed that the nucleus of wounded plant cells often
becomes associated with the cytosolic membrane close to the wound site,
suggesting that the T-DNA could be transferred directly to the nucleus without
extensive exposure to the cytosol.
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