Transfer DNA, or T-DNA, is a segment of DNA from a bacterium with the natural capacity to integrate into the genetic material of plants. This process, where genetic information moves from a bacterium to a plant, is a naturally occurring form of genetic modification. The T-DNA is part of a larger system that facilitates its journey from the bacterium into the nucleus of a plant cell.
The discovery and understanding of its mechanics have provided a foundational platform for modern plant biotechnology. Scientists have learned to harness this natural process, using the T-DNA system as a vehicle to introduce specific, desired genes into plants. This has made it a widely used instrument for developing new crop varieties.
The Source of T-DNA and Crown Gall Disease
The natural origin of T-DNA is the soil bacterium Agrobacterium tumefaciens. This microorganism contains a large, circular piece of DNA separate from its main chromosome, known as a tumor-inducing (Ti) plasmid, which carries the T-DNA segment. In nature, the bacterium uses this T-DNA to cause a plant disease called crown gall.
Crown gall disease manifests as a tumor-like growth, or gall, at the junction of the root and stem. This occurs because the transferred T-DNA carries genes that disrupt the normal regulation of cell growth by coding for enzymes that synthesize plant hormones. The overproduction of these hormones leads to the uncontrolled cell division that forms the gall.
This process serves a purpose for the bacterium. In addition to hormone-producing genes, the T-DNA carries genes that force the plant cell to produce compounds called opines. Opines are amino acid derivatives that the plant cannot use, but they serve as an exclusive source of carbon and nitrogen for the Agrobacterium.
The Molecular Mechanism of Transfer
The transfer of T-DNA from Agrobacterium to a plant cell is initiated by signals from the plant. When a plant is wounded, it releases phenolic compounds into the soil. These chemicals are detected by the bacterium and activate a set of virulence (vir) genes, which are also located on the Ti plasmid but outside the T-DNA region.
Upon activation, the vir genes produce proteins that carry out the transfer. Two of these proteins, VirD1 and VirD2, function as molecular scissors, recognizing and binding to specific 25-base-pair sequences known as the left and right borders that flank the T-DNA. The VirD2 protein cuts the DNA at these borders, excising a single-stranded version of the T-DNA, to which it remains attached.
This single-stranded T-DNA, called the T-strand, must be protected on its journey. The VirD2 protein helps shield one end, while another protein, VirE2, coats the entire length of the T-strand to protect it from being degraded. The resulting combination of the T-strand and its protective protein coat is known as the T-complex.
The T-complex is then transported out of the bacterium and injected into the plant cell through a structure called a Type IV secretion system. Once inside the plant cell cytoplasm, the T-complex is guided toward the nucleus. The proteins in the complex contain signals recognized by the plant’s nuclear import machinery, facilitating entry into the nucleus where the T-DNA integrates into the plant’s DNA.
Applications in Genetic Engineering
The natural ability of Agrobacterium to insert its T-DNA into a plant genome has been adapted for use in biotechnology. Scientists modify the Ti plasmid by “disarming” it, which involves removing the native genes within the T-DNA region responsible for causing tumors and synthesizing opines. The border sequences are left intact, as they are necessary for the transfer process.
With the tumor-causing genes removed, the T-DNA region becomes a blank slate where a “gene of interest” can be inserted. This gene can be any piece of DNA that codes for a desirable trait. The modified Ti plasmid, now carrying a useful gene, is then reintroduced into Agrobacterium.
When this engineered bacterium infects a plant, it transfers the modified T-DNA containing the new gene into the plant’s cells. Because the T-DNA no longer carries the genes for hormone overproduction, no gall is formed. Instead, the plant cell and all the cells that develop from it now carry the new gene and express the desired trait.
This method has been used to develop numerous commercially important crops.
- Insect-resistant cotton and corn have been created by inserting a gene from the bacterium Bacillus thuringiensis (Bt) that produces a protein toxic to certain insect larvae.
- Herbicide-tolerant soybeans and canola have been developed by introducing a gene that allows the plant to withstand specific chemical weed killers.
- Golden Rice was engineered to produce beta-carotene, a precursor to Vitamin A, by inserting genes into the T-DNA to address nutritional deficiencies.