thumb|350px|right|alt=A diagram showing the structure of the Ti plasmid, with various important regions labeled|The structure of the Ti plasmid

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. 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.

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. 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. Therefore, while the complete process behind the replication of the Ti plasmid has not been fully described, the initial step of replication would likely depend on the expression of RepC and its binding to oriV. Of note, the RepC protein only acts in cis, where it only drives the replication of the plasmid it is encoded in and not any other plasmid also present in the bacterial cell. Mutations in either of the proteins RepA or RepB have resulted in a decrease in plasmid stability, indicating their role and importance in plasmid partitioning. RepE is complementary to RepC and will bind with the repC mRNA to form a double-stranded molecule. This can then block the translational production of the RepC protein. This is a type of sensing and signalling system found commonly in bacteria; in this case, they act to sense plant-derived signals to drive the expression of the vir region. During the sensing, VirA, a histidine sensor kinase, will become phosphorylated before passing on this phosphate group to the response regulator VirG. The activated response regulator VirG can then bind to a region of DNA known as the vir box, located upstream of each vir promoter, to activate the expression of the vir region. Furthermore, with the induction of the vir region, the transfer of T-DNA can be mediated by the Vir proteins.

The virB operon is the largest operon in the vir region, encoding for 11 VirB proteins involved in the transfer process of T-DNA and bacterial proteins into host plant cells (see transfer apparatus below).

The virC operon encodes for two proteins: VirC1 and VirC2. These proteins influence the pathogenesis of the Agrobacterium towards different plant hosts, and mutations can reduce but not remove the virulence of the bacteria. Both the virC and virD operons can be repressed by a chromosomally encoded protein known as Ros. Ros binds to a region of DNA that overlaps with the binding site of the VirG regulator, and therefore competes with VirG to control their expression levels. VirD1 and VirD2 are involved in the processing of T-DNA during conjugation to produce the T-strand; this is the single-stranded DNA molecule that is transported to the host plant cell (see transfer apparatus below). During the processing, VirD1 will act as a topoisomerase to unwind the DNA strands. Within the recipient cell, VirD2 will also work together with VirE2 to direct the transferred DNA to the recipient cell's nucleus. There are suggestions that VirD2 may be phosphorylated and dephosphorylated by different proteins, affecting its ability to deliver DNA. Conversely, little is known about VirD3, and mutational analyses have not provided any support for its role in the virulence of Agrobacterium. Finally, VirD4 is a crucial part of the conjugation process, serving as a coupling factor that recognizes and transfers the T-strand to the transport channel.

The virE operon encodes for 2 proteins: VirE1 and VirE2. VirE2 is an effector protein translocated together with the T-strand into host plant cells. There, it binds to the T-strand to direct its delivery to the nucleus of the host plant cell. Part of this activity involves the presence of nuclear localization sequences within the protein, which marks the protein and the associated DNA for entry into the nucleus. It also protects the T-strand from nuclease attack. There is some speculation regarding the role of VirE2 as a protein channel, allowing DNA to move through the plant cytoplasmic membrane. On the other hand, VirE1 may be involved in promoting the transfer of the VirE2 protein into the host plant cell. It binds to the ssDNA-binding domain of VirE2, therefore preventing the VirE2 protein from prematurely binding to the T-strand within the bacterial cell.

virF is a host specificity factor found in some but not all types of Ti plasmids; for example, octopine-type Ti plasmids possess virF but nopaline-types do not. The ability of A. tumefaciens to induce crown gall tumours in certain plant species but not others has been attributed to the presence or absence of this virF gene. A bioinformatics study of the amino acid sequences of the VirH protein showed similarities between them and a superfamily of proteins known as cytochrome P450 enzymes. VirH2 was then discovered to metabolize certain phenolic compounds detected by VirA.

For the Ti plasmid and T-DNA to be transferred via conjugation, they must first be processed by different proteins, such as the relaxase enzyme (an enzyme that nicks DNA) TraA/VirD2 and the DNA transfer and replication (Dtr) proteins. traA and its Dtr recognize and bind to a region known as the origin of transfer (oriT) in the Ti plasmid to form the relaxosome, a protein complex for plasmid transfer. VirD2 and its Dtr instead recognize and create a nick at T-DNA's border sequence, and the nicked T-strand will be transported to the cell membrane, where the rest of the transfer machinery is present.

Within the VirB/VirD4 system, the VirD2 relaxase is aided by the accessory factors VirD1, VirC1 and VirC2 while it processes the DNA substrate. Furthermore, the VirD2 relaxase and the VirC proteins will contribute to the delivery of the DNA strand to the VirD4 receptor at the cell membrane. This receptor is an essential component of T4SSs, and is thought to energize and mediate the transfer of the DNA into the translocation channel between two cells.

The Tra/Trb and VirB/VirD4 systems are evolutionally related and each organized as an operon on the plasmid. The table below summarizes the proteins encoded in the operons that makes up the translocation channel of the two systems.

|-

| VirB2 || TraM || TrbC

| The major pilin subunit of the conjugative pilus.

|-

| VirB4 || TraB || TrbE

| ATPase that provide the energy for pilin dislocation. Is a possible channel subunit. specifically the tip adhesin. Proteins involved in mediating the transfer of T-DNA will first recognize the border sequences of the T-DNA region. Therefore, it is possible for scientists to use T-DNA border sequences to flank any desired sequence of interest - such a product can then be inserted into a plasmid and introduced into Agrobacterium cells. There, the border sequences will be recognized by the transfer apparatus of A. tumefaciens and delivered in a standard manner into the target plant cell. This method has been used to modify several crop plants, including rice, barley and wheat. Further work have since extended the targets of A. tumefaciens to include fungi and human cell lines.

Similar plasmids

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Various RepABC plasmids form incompatibility groups. Plasmids of the same group usually cannot co-exist with one driving another to extinction. However, many nopaline-type Ti plasmids have the ability to fuse with incompatible plasmids. This makes these plasmids hard to "cure" (remove from the bacterium), a process often used in the lab to swap a Ti plasmid for another.

Symbiotic (sym) plasmids of Rhizobia

Some members of Rhizobia carry their nitrogen-fixing genes (nif) and nodulation genes (nod) on the chromosome, while others carry them on specialized symbiotic (sym) plasmids. Like Ti plasmids, the sym plasmids also use the repABC replacation, the TraI/TraR quorum sensing system, and contain a trb plasmid transfer system. A typical example is pNGR234a (536,165&nbsp;bp; accession No. NC000914) from Sinorhizobium fredii NGR234.

See also

  • Mary-Dell Chilton
  • Jeff Schell
  • Marc Van Montagu

References

  • Agrobacterium tumefaciens plasmid pTi-SAKURA, complete sequence
  • Ti Plasmid Genetic Map
  • Crown gall disease