A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of Agrobacterium sensu lato, including A. tumefaciens, Rhizobium rhizogenes, A. rubi and Allorhizobium vitis. Evolutionarily, the Ti plasmid is part of a family of plasmids carried by many species of Alphaproteobacteria. Members of this plasmid family are defined by the presence of a conserved DNA region known as the repABC gene cassette, which mediates the replication of the plasmid, the partitioning of the plasmid into daughter cells during cell division as well as the maintenance of the plasmid at low copy numbers in a cell. The Ti plasmids themselves are sorted into different categories based on the type of molecule, or opine, they allow the bacteria to break down as an energy source. The presence of this Ti plasmid is essential for the bacteria to cause crown gall disease in plants. This is facilitated via certain crucial regions in the Ti plasmid, including the vir region, which encodes for virulence genes, and the transfer DNA (T-DNA) region, which is a section of the Ti plasmid that is transferred via conjugation into host plant cells after an injury site is sensed by the bacteria. These regions have features that allow the delivery of T-DNA into host plant cells, and can modify the host plant cell to cause the synthesis of molecules like plant hormones (e.g. auxins, cytokinins) and opines and the formation of crown gall tumours. Because the T-DNA region of the Ti plasmid can be transferred from bacteria to plant cells, it represented an exciting avenue for the transfer of DNA between kingdoms and spurred large amounts of research on the Ti plasmid and its possible uses in bioengineering.
Nomenclature and classification The Ti plasmid is a member of the RepABC plasmid family found in Alphaproteobacteria. These plasmids are often relatively large in size, ranging from 100kbp to 2Mbp. They are also often termed replicons, as their replication begins at a single site. Members of this family have a characteristic repABC gene cassette. Another notable member of this family is the root inducing (Ri) plasmid carried by A. rhizogenes, which causes another plant disease known as hairy root disease. A key feature of Ti plasmids is their ability to drive the production of opines, which are derivatives of various amino acids or sugar phosphates, in host plant cells. These opines can then be used as a nutrient for the infecting bacteria, which catabolizes the respective opines using genes encoded in the Ti plasmid. Accordingly, Ti plasmids have been classified based on the type of opine they catabolize, namely: nopaline-, octopine- or mannityl-types, which are amino acid derivatives, or agrocinopine-type, which are sugar phosphate derivatives.
Historical discovery The identification of A. tumefaciens as the cause of gall tumours in plants paved the way for insights into the molecular basis of crown gall disease. The first indication of a genetic effect on host plant cells came in 1942-1943, where plant cells of secondary tumours were found to lack any bacterial cells within. However, these tumour cells did possess the ability to produce opines metabolized by the infecting bacterial strain. Crucially, the production of the respective opines occurred regardless of the plant species and occasionally only within crown gall tissues, indicating that the bacteria had transferred some genetic material to the host plant cells in order to allow opine synthesis. However, how and to what extent did DNA transfer occur remained an open question. Adding A. tumefaciens DNA alone did not cause tumors in plants, while very little A. tumefaciens DNA was found to be integrated into the host plant cell genome. The addition of deoxyribonucleases (DNases) to degrade DNA also failed to prevent the formation and growth of the plant tumors. These suggested that little, if any, of the A. tumefaciens DNA is transferred to the host plant cell to cause disease and, if DNA is indeed transferred from the bacteria to the plant, it must occur in a protected manner. Subsequently, oncogenic bacterial strains were found to be able to convert non-pathogenic bacteria into pathogens via the process of conjugation, where the genes responsible for virulence were transferred to the non-pathogenic cells. The role of a plasmid in this pathogenic ability was further supported when large plasmids were found only in pathogenic bacteria but not avirulent bacteria. Eventually, the detection of parts of bacterial plasmids in host plant cells was established, confirming that this was the genetic material responsible for the genetic effect of infection. With the identification of the Ti plasmid, many studies were carried out to determine the characteristics of the Ti plasmid and how the genetic material is transferred from the Agrobacterium to the plant host. Some notable early milestones in the studies of Ti plasmids include the mapping of a Ti plasmid in 1978 and the studying of sequence similarity between different Ti plasmids in 1981. Between 1980–2000, the characterization of the T-DNA region and the 'vir' region was also pursued. Studies into the T-DNA region determined their process of transfer and identified genes allowing the synthesis of plant hormones and opines. Separately, early work aimed to determine the functions of the genes encoded in the 'vir' region - these were broadly categorized into those that allowed bacterial-host interactions and those that enabled T-DNA delivery.
Replication, partitioning and maintenance
The replication, partitioning and maintenance of the Ti plasmid depends on the repABC gene cassette, which is mainly made up of three genes: repA, repB and repC. repA and repB each encode for proteins involved in plasmid partitioning, while repC encodes a replication initiator. These genes are expressed from 4 different promoters located upstream of repA. repE encodes for a small antisense RNA and is located between repB and repC. Additionally, there is a partitioning site (parS) and an origin of replication (oriV) present within the repABC cassette.
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