Ti plasmid

1. Ti plasmid
Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to Bengaluru North University,
K. Narayanapura, Kothanur (PO)
Bengaluru 560077

2. Nature’s genetic engineer
1. Agrobacterium is considered as the nature’s genetic engineer.
2. Agrobacterium tumefaciens is a rod shaped, gram negative bacteria found in
the soil that causes tumorous growth termed as crown gall disease in dicot
plants.
3. The involvement of bacteria in this disease was established by Smith and
Townsend (1907).
4. Agrobacterium contains a transfer DNA (T-DNA) located in its tumor-inducing
(Ti) plasmid that is transferred into the nucleus of an infected plant cell.
5. The T-DNA gets incorporated into the plant genome and is subsequently
transcribed. The T-DNA integrated into the plant genome carries not only
oncogenic genes but also opine synthesizing genes.
Agrobacterium “Species” And Host Range
The genus Agrobacterium has been divided into a number of species on the
basis of symptoms of disease and host range.
A. radiobacter is an “avirulent” species,
A. tumefaciens causes crown gall disease,
A. rubi causes cane gall disease,
A. rhizogenes causes hairy root disease and
A. vitis causes galls on grape and a few other plant species

3. Molecular basis of Agrobacterium-mediated transformation
Ti-plasmid
The virulent strains of A. tumefaciens harbor large plasmids (140–235 kbp) known as
tumor-inducing (Ti) plasmid involving elements like
A. T-DNA – Left border and Right border, Auxin and cytokinin and opine synthesizing
genes
B. vir region- Genes in the virulence region are grouped into the operons vir ABCDEFG,
which code for the enzymes responsible for mediating conjugative transfer of T-DNA to
plant cells.
C. origin of replication,
D. region enabling conjugative transfer and
E. o-cat region (required for catabolism of opines).
•The plasmid has 196 genes that code for 195 proteins.
• The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is
coding genes.
•There are no pseudogenes.

4. •The Ti plasmid is lost when Agrobacterium is grown above 28 °C.
•The modification of this plasmid is very important in the creation of
transgenic plants.
A.) T- DNA (Transfer DNA)
1. It is a small, specific segment of the plasmid, about 24kb in size
and found integrated in the plant nuclear DNA at random site.
This DNA segment is flanked by right and left borders.
2. This region has the genes for the biosynthesis of auxin (aux),
cytokinin (cyt) and opine (ocs), and is flanked by left and right
borders. It is clearly established that the right border is more
critical for T -DNA transfer.
3. The T-DNA contains two groups of genes, which possess the
ability to express in plants as follows-
i. Oncogenes for synthesis of auxins and cytokinins
(phytohormones). The overproduction of phytohormones leads
to proliferation of callus or tumour formation in plants.
ii. Opine synthesizing genes for the synthesis of opines. Thus
opines act as source of nutrient for bacterial growth, e.g.
Octopine, Nopaline.

5. The functions of T-DNA genes are listed:

6. B) Virulence genes (vir genes)
1. Virulence genes aid in the transfer of T-DNA into the host plant cell.
2. Ti plasmid contains 35 vir genes arranged in 8 operons.
3. At least nine vir-gene operons have been identified. These include vir A, vir G, vir B1, vir
C1, vir D1, D2, vir D4 and vir E1, E2.
4. Transfer the T-DNA to plant cell
5. Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes.
6. virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about
30 kb of Ti plasmid.
Function of vir genes
virA - transports acetosyringone (AS) into bacterium, activates virG post-translationally (by
phosphorylation)
virG - promotes transcription of other vir genes
virD2- endonuclease/integrase that cuts T-DNA at the borders but only on one strand.
virE2 - can form channels in membranes
virE1 - chaperone for virE2
virD2 & virE2 also have NLSs, gets T-DNA to the nucleus of plant cell
virB - operon of 11 proteins, gets T-DNA through bacterial membranes

7. C) Opines:
Derivatives of amino acids synthesized by T-DNA.
This region codes for proteins involved in the uptake and metabolisms of opines.
Ti plasmids can be classified according to the opines produced :
1. Nopaline plasmids - carry gene for synthesizing nopaline in the plant and for
utilization (catabolism) inthe bacteria.
2. Octopine plasmids - carry genes to synthesize octopine in the plant and catabolism in
the bacteria.
3. Agropine plasmids - carry genes for agropine synthesis and catabolism.
Opine genes can be used as marker genes-
For screening of transformants or
recombinants

9. DNA transfer into the plant genome
Ti plasmid
Ti Plasmid

10. Ti plasmid based vectors:
1. Disarmed Ti-plasmid derivatives as plant vectors
2. Binary Vectors
3. C0-integrate vectors
1. Disarmed Ti vector
1. Ti plasmid is a natural vector for genetically engineering plant cells due to its ability to
transfer T-DNA from the bacterium to the plant genome.
2. But wild-type Ti plasmids are not suitable as vectors due to the presence of oncogenes
in T-DNA that cause tumor growth in the recipient plant cells.
3. For efficient plant regeneration, vectors with disarmed T-DNA are used by making it
non-oncogenic by deleting all of its oncogenes.
4. The foreign DNA is inserted between the RB and LB and then integrated into the plant
genome without causing tumors.

11. Construction: Structure of the Ti-plasmid pGV3850 with disarmed
T-DNA
1. Zambryski et al. (1983) substituted pBR322 sequences in the T-DNA of pTiC58, without
disturbing the left and right border regions and the nos gene.
2. The resulting construct was called pGV3850. No tumour cell formation takes place
when modified T-DNA is transferred from Agrobacterium carrying pGV3850 plasmid.
3. The evidence of transfer is done by screening the cells for nopaline production.
Drawbacks
Several drawbacks are associated with
disarmed Ti- vector systems are:
• Necessity to carry out enzymatic assays on
all potential transformants.
• Not convenient as experimental gene
vectors due to large size.
• Difficulty in in vitro manipulation and
• Absence of unique restriction sites in the
T-DNA.

12. 2. Binary vector
• Binary vector was developed by Hoekma et al (1983) and Bevan in (1984).
• It utilizes the trans- acting functions of the vir genes of the Ti-plasmid and can act on any T-
DNA sequence present in the same cell.
• Binary vector contains transfer apparatus-helper plasmid (contains the vir genes) and the
disarmed T-DNA containing the transgene on separate plasmids.
Binary vector system
Binary vector consists of a pair of plasmids:
1) A disarmed Ti plasmid: This plasmid has T-DNA containing LB and RB with gene of interest +
ori for both E. coli and Agrobacterium. Also called as mini-Ti or micro Ti plasmid eg: Bin 19.
2) Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti plasmid to
the plant.
Plasmid with disarmed
T-DNA but without
vir gene
Plasmid with vir region but
without G0I

13. A binary vector system
Plasmid with disarmed T-
DNA but without vir gene
Plasmid with vir region but
without G0I

14. Binary vector Cloning Strategy:
1. Move T-DNA onto a separate, small
plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin
resistance) gene in T-DNA.
4. Vir genes are retained on a separate
plasmid.
5. Put foreign gene between T-DNA
borders.
6. Co-transform Agrobacterium with both
plasmids.
7. Infect plant with the transformed
bacteria.
Examples of Binary vector system
pBIN19- one of the first binary vectors developed in 1980s and was widely used.
pGreen- A newly developed vector with advanced features than pBIN19.

15. 3. Co- integrate vectors
Transfer is achieved using a ‘triparental mating’ in which three bacterial strains are mixed
together:
(i) An E. coli strain carrying a helper plasmid able to mobilize the intermediate vector in
trans;
(ii) The E. coli strain carrying the recombinant intermediate vector;
(iii) A. tumefaciens carrying the Ti plasmid
1. In first E.coli strain: The DNA to be introduced into the plant transformation vector is
sub cloned in a conventional Escherichia coli plasmid vector for easy manipulation,
producing a so-called intermediate vector. These vectors are incapable of replication
in A. tumefaciens and also lack conjugation functions.
2. --- An another E. coli strain carrying a helper plasmid able to mobilize the
intermediate vector .
3. Conjugation between the two E. coli strains transfers the helper plasmid to the
carrier of the intermediate vector, which in turn is mobilized and transferred to the
recipient Agrobacterium.
4. Homologous recombination between the T-DNA sequences of the Ti plasmid and
intermediate vector forms a large co- integrate plasmid resulting in the transfer of
recombinant T-DNA to the plant genome.

16. Construction of a Co-integrate vector (foreign gene cloned into an appropriate plasmid is
integrated with a disarmed Ti-plasmid through homologous recombination).
1. At first; an intermediate vector is made using E. coli plasmid + origin of replication +
pBR322 sequences + some markers + gene of interest.
2. -Second vector is a disarmed pTi vector = Left and right borders+ some markers +
pBR322 sequences + vir region.
3. -Both intermediate vector and disarmed pTi has some sequences in common (pBR322
sequences).
4. -Therefore by homologous recombination, co-integration of two plasmids will take
place within Agrobacterium.
5. Now we have a co-integrate vector that has both T-DNA with our gene of interest with
in the T-DNA borders and vir region. This complete vector is used for transformation in
plant cells. eg: pGV2260.
in
Agrobac
terium
tumefac
iens