Ti plasmid as vector for plants

• 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

1. Octopine
2. Nopaline
3. Succinamopine
4. 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.

1. 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.
2. virB encodes proteins which produce a pore/pilus-like structure.
3. virC binds the overdrive sequence.
4. virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA segment, beginning with the right border.
5. 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.