Monday, 30 July 2012

Ti plasmid

Ti Plasmid

What is Ti Plasmid:

-          Ti plasmid (stands for tumor inducing) is a circular plasmid of Agrobacterium tumefaciens. pTi and pRi (plasmid of Agrobacterium rizogenes, responsible for hairy root production)  share little sequence homology but are functionally rather similar.
-          The Ti plasmids are classified into different types based on the type of opine produced by their genes. The different opines specified by pTi are octopine, nopaline, succinamopine and leucinopine.
-          The plasmid has 196 genes that code for 195 proteins.
-          There is no one structural RNA.
-          Size of the plasmid: ~250 kbp and 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding genes.
-          There are no pseudogenes.
-          Importance: Agrobacterium is called the natural genetic engineer. Very important in the creation of transgenic plants, but only in dicotyledon plants.

Structure of Ti Plasmid:

-          Contains one or more T-DNA region.
-          Contains a region enabling conjugative transfer.
-          Contains regions for opine synthesis and catabolism.
-          Responsible for crown gall disease in plants.

Condition for Ti Plasmid transfer:

- The Ti plasmid is lost when Agrobacterium is grown above 28°C. Such cured bacteria do not induce crown galls, i.e. they become avirulent.
- A. tumefaciens it was observed that the attachment of wild-type bacterium to plant cells was directly correlated with the production of an acidic polysaccharide.
- The activation of vir system also depends on external factors like temperature and pH. At temperatures greater than 32°C, the vir genes are not expressed because of a conformational change in the folding of VirA induce the inactivation of its properties. The effect of temperature on VirA is suppressed by a mutant form of VirG (VirGc), which activates the constitutive expression of the vir genes. How ever, this mutant cannot confers the virulence capacity at that temperature to Agrobacterium, probably because the folding of other proteins that actively participate in the T-DNA transfer process are also affected at high temperature


Production of transgenic plants by use of co-integrated Ti plasmids.

    The soil bacterium Agrobacterium tumefaciens causes crown gall disease in plants by transferring the T-DNA region of a tumor-inducing (Ti) plasmid into host cells (Top). The T-DNA region of the Ti plasmid can be genetically engineered to contain an antiobiotic resistance gene (kanR) as well as a foreign gene of interest (inset diagram). Infection of plant cells in culture with bacteria containing this co-integrated Ti plasmid allow the foreign DNA to be transferred into the host cell.  Integration of the foreign DNA disrupts tumor formation, and only those plant cells with the kanR gene will grow in culture containing antibiotic. Plants are easily regenerated from cultured cells (calluses): the adult transgenic plant expresses the foreign gene.

Gene responsible for Ti Plasmid Transfer

T-DNA contains two types of genes: the oncogenic genes, encoding for enzymes involved in the synthesis of auxins and cytokinins and responsible for tumor formation; and the genes encoding for the synthesis of opines. These compounds, produced by condensation between amino acids and sugars, are synthesized and excreted by the crown gall cells and consumed by A. tumefaciens as carbon and nitrogen sources. Outside the T-DNA are located the genes for the opine catabolism, the genes involved in the process of TDNA transfer from the bacterium to the plant cell and the genes involved in bacterium-bacterium plasmid conjugative transfer.
Different chromosomal-determined genetic elements have shown their functional role in the attachment of A. tumefaciens to the plant cell and bacterial colonization: the loci chvA and chvB, involved in the synthesis and excretion of the b-1,2 glucan; the chvE required for the sugar enhancement of vir genes induction and bacterial chemotaxis; the cel locus, responsible for the synthesis of cellulose fibrils; the pscA (exoC) locus, playing its role in the synthesis of both cyclic glucan and acid succinoglycan; and the att locus, which is involved in the cell surface proteins.
 Genes in the virulence region are grouped into the operons virABCDEFG, which code for the enzymes responsible for mediating transduction of T-DNA to plant cells.
Vir Genes and their FunctionVir Gene     
Vir A, Vir G-
Sense phenolic compounds from wounded plant cells such as acetosyringone, syringealdehyde or acetovanillone which leak out of damaged plant tissues and induce expression of other virulence genes. Activation of VirA by these phenolic inducers initiates a phospho-relay, ultimately resulting in phosphorylation and activation of the VirG protein. Activated VirG binds to the vir box sequences preceding each vir gene operon, allowing increased expression of each of these operons. In addition to induction of the vir genes by phenolics, many sugars serve as co-inducers. These sugars are perceived by a protein, ChvE, encoded by a gene on the Agrobacterium chromosome. In the presence of these sugars, vir genes are more fully induced at lower phenolic concentrations. VirA protein can be structurally defined into three domains: the periplasmic or input domain and two transmembrane domains (TM1 and TM2). The TM1 and TM2 domains act as a transmitter (signaling) and receiver. The TM1 and TM2 domains act as a transmitter (signaling) and receiver (sensor). The periplasmic domain is important for monosaccharide detection. Within the periplasmic domain, adjacent to the TM2 domain is an amphipatic helix, with strong hydrophilic and hydrophobic regions. This structure is characteristic for other transmembrane sensor proteins and folds the protein to be simultaneously aligned with the inner membrane and anchored in the membrane. The TM2 is the kinase domain and plays a crucial role in the activation of VirA, phosphorylating itself on a conserved His-474 residue in response to signaling molecules from wounded plant sites. Monosaccharide detection by VirA is an important amplification system and responds to low levels of phenolic compounds. The induction of this system is only possible through the periplasmic sugar (glucose/galactose) binding protein ChvE, which interacts with VirA. Recent studies for determination of VirA regions, important for its sensing activity suggested the position, which may be involved on TM1-TM2 interaction . This interaction causes the exposure of the amphipathic helix to small phenolic compounds and suggests a putative model for the VirAChvE interaction.
virB-  Encodes proteins which produce a pore/pilus-like structure
VirD2- Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis
Vir D1-Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp border sequence
Vir D2-Covalently attaches to the 5I end of the T-strand, thus forming the T-DNA Complex. Also guides the T-DNA complex through the nuclear pores
Vir C- Binds to the 'overdrive' region to promote high efficiency T-strand Synthesis
Vir E2-Binds to T-strand protecting it from nuclease attack, and intercalates with lipids to form channels in the plant membranes through which the  T-complex passes
Vir E1- Acts as a chaperone which stabilises Vir E2 in the Agrobacterium
Vir B & Vir D4- Assemble into a secretion system which spans the inner and outer bacterial membranes. Required for Export of the T-complex and Vir E2 into the plant cell


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