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Tumor Inducing Plasmids (Ti plasmid) of Agrobacterium
#1
Tumor inducing plasmids (Ti Plasmids) are double stranded circular DNA present in Agrobacterium tumefaciens. This article gives you complete information of these Ti Plasmids.

Agrobacterium is a gram negative soil bacterium which infects over 3000 dicots and causes crown gall disease at the collar region. This plasmid is denatured at higher temperatures and loses tumorgenic properties. Ti plasmid encode for enzymes for catabolism of opines such as permease and oxidase.

Ti plasmid ranges from 180-205kb in size. It has T DNA which is of 20kb and in addition it has several genes such as vir genes for virulence, ori gene for origin of replication, tra genes for transfer and genes for opine synthesis. Virulence genes are responsible for the transfer of T DNA into the host cell and integration of T DNA with host genome. Opines are derivatives of amino acids which are of two types; octopine and nopaline. Octopine is formed with two amino acids; Arginine and Alanine. Nopaline is made up of Arginine and Glutamine. Octopine and nopaline are not found in healthy plant tissues. The opines are catabolized and used as the energy source by the bacterium. A. tumefaciens is able to divert the metabolic resources of the host plant to the synthesis of opines which are of no apparent benefit to the plant. But they provide sustenance to the bacterium. During the infection through a wound, the plant cells begin to proliferate and form tumors and the plant tissues begin to synthesize opines.

Ti plasmids are classified based on the type of opines they produce in the host cell during infection. Almost all Ti plasmids have identical structure except in their sequence for opine metabolism. Octopine Ti plasmids produce Octopine ( C9H18N4O4). Tra genes encode proteins necessary for transfer of T DNA into the host. The Inc locus in octopine plasmids causes incompatibility of the plasmid in the bacterium. Shi and Roi sequences regulates shoot induction and root induction respectively. Nopaline Ti plasmids produces an opine called as nopaline ( C9H16N4O6).

T DNA is region of Ti plasmids common to both octopine and nopaline plasmids. But generally T DNA segment of Octopine plasmids is shorter than in Nopaline plasmids. In some octipine Ti plasmids, T DNA occurs in two segments. The left segment with 13kb in length and contains information for the synthesis of growth hormones and a sequence for opine synthesis. The right segment does not participate in tumor formation and maintenance.

Ti plasmids have a single repeated sequence at both ends of T DNA. This sequence is called as the bordered sequence. These two sequences act as sites during the transfer of Ti plasmid into the plant genome. It is found that removal of left bordered sequence doesn’t cause considerable change in tumor induction in the infected tissues. But the removal of right border sequence from T DNA results in failure of tumor induction. T DNA encode for the production of growth hormones like Cytokinin and Auxin which is necessary for tumor induction.
Vir region contains eight operons; Vir A, Vir B, Vir C, Vir D, Vir E, Vir F, Vir G and Vir H which together span about 40kb of DNA. This region mediates the transfer of T DNA into plant genome. The genes of vir region are not transferred by themselves, they only induce the transfer. The bordered sequences are essential for the transfer. Vir A and Vir G genes regulate Vir operon. Vir region is activated by the phenolic compounds, namely Acetosyringone and alpha hydroxy acetosyringone produces by plant wounds. These bind to Vir A proteins activates Vir G gene by phosphorylation which in turn activates other genes. Vir D proteins together with Vir D proteins participate in the formation of conjugal tube formation between the bacterial cell and plant tissue during the infection process.

In transferring gene of interest to Ti plasmid, an intermediate vector is used such as pBR 322. T DNA portion of the Ti plasmid is separated. T DNA is inserted into the pBR 322 vector which results in formation of a shuttle vector. This shuttle vector can replicate in E. coli and in Agrobacterium.

Ti plasmids serve as ideal vectors for plant genetic engineering as it has the capability of transferring gene of interest into the target site with a high efficiency. It is easy to screen recombinant cells as marker genes are present.
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#2
thank you so much ishani Smile i found it very useful Smile i was surfing the net hours togethr to knw about ti-plasmid but nothing helped me. atlast, i was like yaay!!! i found it!!! Big Grin
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#3
Plant cells do not have any endogenous plasmids. The plasmid vectors used for plant cell transformation are mostly based on pTi (tumor inducing plasmid) of A.tumifaciens, but some are derived from pRi (root inducing plasmid) of A.rhizogenes. these are plant pathogenic, gram negative soil bacteria known for more than 90 years to cause crown gall (A.tumifaciens) and hairy root (A.rhizogenes) disease of dicot plants. They infect plant cells near wounds, usually at the crown of roots at the soil surface.

Mechanism of Agrobacterium Infection:

The molecular mechanisms involved in Agrobacterium infection of plant cells became known only recently during the 1980s. the process of infection by A.tumifaciens culminates in the transfer of a small part of pTi into the plant cell genome; this DNA sequence is called T-DNA (transferred DNA). The infection process is governed by both chromosomal and the plasmid-borne genes of A.tumifaciens. Infection begins when Agrobacterium cells become attached to plant cells; this step determines the host range of bacterium, is a function of host –parasite interaction, and is governed by bacterial chromosomal genes, generally the chv (chromosomal virulence) genes. Most of these genes, e.g., chvB, exo genes, cel genes, are concerned with the biosynthesis of cell attachment polysaccharides due to which the bacterial cells become firmly adhered to plant cells. But at least two genes, viz., chvD and chvE, are needed for an optimal expression of pTi vir genes. These chromosomal genes are expressed constitutively, i.e. expressed in all bacterial cells at all the times.

Vectors derived from pTi:

The use of wild type pTi as a vector presents the following three problems:
1. presence of oncogenes (iaaM, iaaH and ipt) in T DNA which causes a disorganized growth and a loss of regeneration potential of the cells having T DNA in their genomes.
2. Their large size makes the handling procedures during cloning tedious and cumbersome.
3. A general lack of unique cloning sites within the T DNA, which are needed for the insertion of DNA segments to be cloned.

These problems have been resolved by deleting the oncogenes from the T-DNA (disarming and by developing intermediate vectors and binary vectors to facilitate gene cloning procedures.
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#4
In addition, a large enough number of recombinant clones are produced so that there is a reasonable chance for the DNA fragments representing each gene to be cloned in all six possible reading frames; three reading frames in each of the two orientations of the fragments with respect to the promoter (present in the vector).
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#5
great value. thanks for sharing.
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Tumor Inducing Plasmids (Ti plasmid) of Agrobacterium00