Introduction
- Agrobacterium species harboring tumor-inducing (Ti) or hairy root-inducing (Ri) plasmids cause crown gall or hairy root diseases, respectively in plants.
- Agrobacterium tumefaciens is a plant pathogen that induces tumor on a wide variety of dicotyledonous plants and the disease is caused by tumor-inducing plasmid (pTi).
- Similarly Agrobacterium rhizogenes is a plant pathogen that induces hairy roots on a wide variety of dicotyledonous plants and the disease is caused by root-inducing plasmid (pRi).
- Virulence (vir) genes of Ri as well as of Ti plasmids are essential for the T-DNA transfer into plant chromosomes .
- These natural plasmids provide the basis for vectors to make transgenic plants.
- The plasmids are approximately 200 kbp in size.
- Both pTi and pRi are unique in two respects:
- (i) they contain some genes, located within their T-DNA, which have regulatory sequences recognized by plant cells, while their remaining genes have prokaryotic regulatory sequences,
- (ii) both plasmids naturally transfer a part of their DNA, the T-DNA, into the host genome, which makes Agrobacterium a natural genetic engineer.
- Complete sequence analysis confirms that the pathogenic plasmids contain gene clusters for DNA replication, virulence, T-DNA, opine utilization and conjugation.
- T-DNA genes have lower G + C content, which is presumably suitable for expression in host plant cells. Besides these genes, each plasmid has a large number of unique genes.
The Ti plasmid
- Agrobacterium tumefaciens is a soil-dwelling bacterium that can transfer part of its Ti (tumor-inducing) plasmid, known as T-DNA, into plant cells, causing crown gall disease.
- The transferred T-DNA integrates into the plant genome and causes infected cells to grow uncontrollably by producing plant growth hormones auxin and cytokinin.
- Wild-type Ti plasmids cannot be used directly for plant genetic engineering because they cause tumor formation. They need to be disarmed by removing the oncogenes while retaining the ability to transfer T-DNA.
The Ti plasmid contains all the genes which required for tumor formation. Virulence genes (vir-genes) are also located on the Ti plasmid. The vir genes encode a set of proteins responsible for the excision, transfer and integration of the T-DNA into the plant nuclear genome.
The basic elements of the vectors designed for Agrobacterium-mediated transformation that were taken from the native Ti-plasmid
- The T-DNA border sequences, at least the right border, which initiates the integration of the T-DNA region into the plant genome.
- The vir genes , which are required for transfer of the T-DNA region to the plant, and
- A modified T-DNA region of the Ti plasmid, in which the genes responsible for tumor formation are removed by genetic engineering and replaced by foreign genes of diverse origin, e.g., from plants, bacteria, virus.
- When these genes are removed, transformed plant tissues or cells regenerate into normal-appearing plants and, in most cases, fertile plants.
The T-DNA region genes are responsible for the tumorigenic process. Some of them control the production of plant growth hormones that cause proliferation of the transformed plant cells. The T-DNA region is flanked at both ends by 24 base pairs (bp) direct repeat border sequence called T-DNA borders. The T-DNA left border is not essential, but the right border is indispensable for T-DNA transfer.
Ti plasmid is grouped into two general categories:
- Nopaline type pTi
- Octopine type pTi