Ti plasmid vector

An agrobacterium based vector to introduce plant genomic fragments., very much used to produce genetic modified plants Pla

2. Introduction
•Ti plasmid is of natural occurrence in in a soil
bacteria –Agrobacteriumtumefaciensinfecting
dicotplant plantcell and induced tumour
formation ( Crown Gall)
•Entry of this bacteria is facilitaedby secretion of
some phenoliccompound from wounded tissues
like Acetosyringone,hydroxyacetosyringone
T DNA is introduced in to plant cell , get integrated in the
plant cell chromosomes and induces un controlled growth

3. Analogous to pUC19 with enormous size of 200kb as compared to pUC19 of
size 4.9 kb
Size -200 kb , independent , extra chromosomal, circular DNA
T DNA -12-24 Kb
Ti plasmid DNA has three regions
T DNA –contains gene for biosynthesis of auxin, opine and cytokiine ( CYT)
and opine (ocs)and flanked by left & rt boarder.
T DNA boarders -a 24 kb on eithe side of Lt & Rt of T DNA
Rt is more critical for Transfer
2. Virulence Regions-gene for promoting transfer of T DNA and located
outside T DNA, is vir ( Virulene regions)-~ 40 kb at least 8~11 vir genes
Nine vir gene, -vir A, G, B1, C1, D1, D2 D4 E1 & E2
Opine catabolism regions –encode a protein helping uptake and metabolism
of opine to provide C & N
Ori region –replication
After infection T DNA is excised and get integrated with chromosomal DNA by
homologous recombination.

4. Classification -On the basis of Opines
• Nopaline plasmids : carry gene for synthesizing
nopaline in the plant and for utilization
(catabolism) in the bacteria.
•Octopine plasmids : carry genes to synthesize
octopine in the plant and catabolism in the
bacteria.
•Agropine plasmids : carry genes for agropine
synthesis and catabolism.

5. Genetic Map
30 kb
a o7 kb a 14 kb
Two component of Ti plasmid are responsible for transformation
1.T DNA
Vir region
T Dna integrates at random site or Multiple sits

6. Structure of T DNA
30 Kb

7. GENE WITHIN T DNA
Gene Product Function
ocs Octopinesynthetase Opine synthesis
nos Nopalinesynthetase Opine synthesis
Tms1Tryptophan 2 mono
oxygenase
Auxin
Tms2 Indoleacetamidehydrolaseauxine
tmr Isopentyltransferase cytokinin
ags Agropinesynthetase cytokinin
Opine –condensation product of either aa & keto acids or
aa and a sugar which act as carbon & nitrogen source

8. Nopaline
Plasmid
Lt & Rt has imperfect terminal repeats and rt removal blocks transfer of T DNA , vir
gene for enzymes needed in excision , integration and transfer during
transformation. Located either cis or in trans positiion T DNA express in very low
levelsin bacteria grwing in soil but in plant increase.within 10-15 hrs
acetosyringosine act as inducer of vir gene

9. The process of transfer and integration is mediated by
Signal transduction –The release chemical acts as signal
molecules to attract Agrobacterium
Attachment–Cellulosic fibres of bacteria helps in
attachment. For this chv ( Chromosomal virulence gene of
bacterial cell to plant cell
Production of virulence protein –a series of event follows
the attachment resultin production of virulence protein
At first vir A expresses which in turn activate vir G and the
DI, D2, E2 B etc are expressed
Some sugars like glucose , galactose , xylulose act as
stimulator.

10. Transfer
Lt & Rtboarder of T DNA are recognisedby virD /virD2 protein for the
production of ssTDNA ,its production and exporttoplant cell The SST
DNA gets attached to virD2
SS T DNA –virD2 complex in association with virG is exported
from bacterial cell . VirB forms transport apparuts
Transfer of T DNA and integration
TDNA –vir D@ complex cross plasma membrane . Complex interact with
vor E2 protein whch prevent T DNA degradation from nuclease
Interaction of vir D2 and E2 and other proteins inflence transport and
integration.
The T DNA –vir D2 vir E2 complex enters through nuclear pore complex
and by recombination integrates

11. Function of virgenes
• virA-transports acetosyringoneinto bacterium,
activates virGpost-translationally(by
phosphorylation)
• virG-promotes transcription of other virgenes
• virD2-endonuclease/integrasethat 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

12. Virgene
Protein products needed for T DNA transfer by
acting as certain receptor for the ligands
secreted by wounded sites in plant . It is neve
trasfeerd in to plant
Vir A-H ( eight types ) and has separate operon
Excision , transfer and translocation in the nucleus

13. Signal transduction
•Acetosyringoneinteracts with VirA and auto
Phosphorylteat Histindine
•Transfer of P to aspartateof virG which act as TF for other V
genes
•Activated virG binds to enhancer (12 bp) present within promotor
of virA,B,C D E operons
•Expression generates SS T DNA
•VirD1 2 act as endonucleaseand cut at 5’sit of T boarder while
cutting it remains attach to prevent exonucleoticactivity
•Excised ssT DNA is transferred and with the help of virE and D get
translocated in to the nucleus as they have NLS
•SST DNA converted into Ds T DNA
•By recombination it gets integrated into the plant genome for
transformation

14. Transfer Mechanism

15. Overview of Infection Process

16. NeopalineTi plasmid is important
•synthesis enzymes for production of Auxin
(IAA) & Cytokine Isopentyl adenosine. For
growth production .
•Antibiotic marker for selection Kanamycin,and
the realted aminoglycoside G418
•Hygromycin and to the drug
•methotrexate

17. Ti plasmid as vector
This ability wass consideed to craete vector to transfer the desired gene for
for transformation of plant cell
Limitation mto be used as vector
Ti Plasmid are large so to be used as vector it
has reduce its size
Absence of RE so , it has to be incorporated
Phytoharmones and auxin synthesising gen
have to remove.
Opine production lowers the transformed yield
so has to be tremoved

18. Ti p as vector
•T DNA is transfer so it is replaced by desired gene to
be considedas vector in transforminplant cell.
•F If it is disarmed i.eremoved Rt& ltdonotcause
tumourso transformed can not be identifed
•For this a marker kanamycinR e.colitransposanTn5
from is added with disarmed T I plasmid / This forms
neomycin phosphotransferasetype II ( NPTII which
detoxify the kanamycinby phosphorylation
•Since it is bacterial specofictherefore can not express
in plant so plant specific protein is ,termination and
poly adenyaltionsequece. Such marker I s calleed
chimericselectable marker

19. •Important points:
•Monocots don't produce AS in response to
wounding.
•Put any DNA between the LB and RB of T-DNA it
will be transferred to plant cell.
•Engineering plants with Agrobacterium: Two
problems had to be overcome:
•Ti plasmids large, difficult to manipulate
•couldn't regenerate plants from tumors

20. Binary vector system
•Strategy:
•Move T-DNA onto a separate, small plasmid.
Remove aux and cytgenes.
•Insert selectable marker (kanamycinresistance)
gene in T-DNA.
•Virgenes are retained on a separate plasmid.
•Put foreign gene between T-DNA borders.
•Co-transform Agrobacteriumwith both plasmids.
Infect plant with the transformed bacteria.

21. Practical application
•Practical application of Agrobacterium-mediated
plant transformation:
•Agrobacteriummediated transformation
methods are thought to induce less
rearrangement of the transgene.
•Lower transgenecopy number that direct DNA
delivery methods.
•Successful production of transgenic plants
depends on the suitable transformation
protocols.

22. Process of Transformation
1
•Explants ( cotyledon, Hypocotyls , microspore
2
•Co-cultivation to allow infection with agrobacteriumcontaingTi vector
•Careendilineto kill bacteria
3
•Transformed or nontransformed
•Selective medium to kill non transformed 9 addition of kanamycin
4•Transformed shoots
5•Rooted shoots
6•Adult plant

23. Conclusion:
•Agrobacteria are biological vector for
introduction of genes into plants.
Agrobacterium-mediated transformation is
not restricted to eukaryotes as Agrobacterium
is also able to act on the gram positive
bacterium Streptomyces lividans.
Agrobacterium can transfer not only DNA but
also proteins to the host organisms through its
type four secretion system.