The document summarizes the mechanism of T-DNA transfer during Agrobacterium tumefaciens infection. It explains that T-DNA is a fragment of DNA transferred from the tumor-inducing (Ti) plasmid of A. tumefaciens into the host plant genome. The T-DNA is bordered by repeats and encodes genes that cause tumors in the plant. Virulence genes are expressed in response to plant signals and produce single-stranded T-DNA, which forms a complex with other proteins and is transported into the plant cell and integrated into the plant nuclear DNA, causing uncontrolled cell growth and tumor formation. The mechanism involves multiple virulence protein complexes and integration of T-DNA is directed by the

1. MECHANISM OF T
DNA
By NIZIGIYUMUREMYI Steev Marcial
II BSc Biotechnology

2. WHAT IS T-DNA?
 T DNA stands for transfer DNA
 It derives its name from the fact that the bacterium transfers
this DNA fragment into the host plant's nuclear DNA
genome.
 It is a part of Ti plasmid (Tumor inducer plasmid) of the
bacillus Agrobacterium tumefaciens
 Agrobacterium tumefaciens is a phytopathogenic, gram-
negative, non sporing, motile bacillus.
 It is found in the rhizosphere
 It causes crown gall disease in plants, a disease of
dicotyledonous plants characterized by a tumorous
phenotype.

3. FEATURES OF TI PLASMID
 It is responsible for crown gall disease
 It has 200 kb
 It has 8 genes (vir, shi, roi, nos, noc, ocs,
occ and ori V)
 A part of Ti-plasmid, T DNA, integrates
into the plant chromosomal DNA
 The T-DNA is bordered by 25-base-pair
repeats on each end. Transfer is initiated at
the right border and terminated at the left
border and requires the vir genes of the Ti
plasmid.

4. MECHANISM OF T DNA
A.Tumefaciens is attracted towards wound site by chemotaxis
The wound induces the production of phenolic compounds such as
acetosyringone
The lipopolysaccharides aid in attachment of A. Tumefaciens.
Ti plasmid code for chemotactic receptors
The processing of T-DNA transfer is mediated by the vir (virulence) region.
The vir gene encode a set of proteins responsible for the excision, transfer and
integration of the T-DNA into the plant nuclear genome: VirB, VirC, VirD,
VirE. Vir A and vir G proteins constitute a two component regulatory system
that controls vir gene expression.

5. MECHANISM OF T DNA
VirA detects the phenolic compounds, acetosyringone, resulting in
autophosphorylation.
The autophosphorylated VirA phosphorylates VirG which leads to the
activation of vir gene and this one get expressed.
 After vir gene activation, T strand, a single stranded copy of T DNA is
produced:
 Two VirD proteins, i.e VirD1 &VirD2, recognize the 25 bp border sequence and
produce a ss endonucleolytic cleavage in the bottom end of each border

6.  These nicks are used as initiation and termination sites for T strand
production
 VirC locus then produces 2 proteins, VirC1 and VirC2 which promotes
generation of multiple copies per cell of T strand.
 Then VirD1 is detached from T strand whereas VirD2 remains tightly
bound on the 5’end to ensure that in subsequent steps, the 5’ end is the
leading end.
 To prevent T strand degradation by endonucleases during its journey to
the plant nucleus, VirE locus produce VirE2 ss nucleic acid-binding
protein

7. MECHANISM OF T DNA
It binds tightly on T-strand which results in the unfolding of the
ssDNA, i.e T-strand, thus reducing its diameter to 2nm
facilitating its transfer through membrane channels.
The T-strand along with VirD2 & VirE2 is called as T complex
The T complex exit the bacterial cell membranes and cross the
plant cell membranes and enter the plant cell.
Once inside the cell, T complex targets the plant nucleus and
cross the nuclear membrane where it integrates into the plant
DNA.

8. MECHANISM OF T DNA

9. The transport of T Complex is mediated by VirB-VirD4 transporter
complex.
Once inside the plant, T complex is led by VirD2 and VirE2
interacting proteins along with VirF towards the nucleus.
In the nucleus, VirF with the interacting proteins unbind from the
t complex
Then the right border initiates integration of tDNA region while
VirD2 and VirE2 are unbinding from the T strand.

10. EFFECT OF T-DNA ON THE PLANT
 T-DNA disorganize the proliferation of cells in the plant
 It reprograms the cell to grow into a tumor and produce a unique source food for the
bacteria
 The
synthesis of the plant hormones auxin and cytokinin enables the plant cell to grow unc
ontrollably, thus forming the crown gall tumors typically.
 The morphology of the galls is controlled by the loci in T-DNA:
 Tml causes large tumors
 Tmr induces tumor in roots
 Tms induces tumor in shoots

11. APPLICATIONS OF T DNA
T-DNA transfer capabilities are used in genetic engineering to introduce
foreign genes into desired plants: the tumor-promoting and opine-
synthesis genes of T-DNA are removed and replaced by genes of interest.
Such plants are then called transgenic plants.

13. Thank you for
your attention!!!