Indirect method of gene transfer

1. In-directMethod
ofGene
Transfermation
Presented by: Manisha Chaudhary
Department of Genetics and plant breeding
Roll no.: PLB-03M-2022
Registration no.:PLB-06M-2021

2. TABLEOFCONTENTS
INTRODUCTION IN-DIRECTMETHODOF
TRANSFORMATION
AGROBACTERIUMMEDIATED
GENETRANSFER
THEPROCESSOFT-DNATRANSFER
ANDINTEGRATION
01 02
04
03

3. 01 INTRODUCTION

4. INTRODUCTION
 Transformation is the process by which genetic makeup of an organism is
altered by the insertion of new gene into its genome
 The aim of producing transgenic plants are
 To improve crop yields
 Improvement of varietal traits
 To make plants more resistance against pest, parasites, pathogens etc.

5. Methodofgenetransformation
Direct Method
IndirectMethod
• Agrobacterium tumefaciens
01
02
Methodofgene
transformation
• Electroporation
• Microprojectile
• Particle gun
• Polyethylene Glycol (PEG)/
protoplast fusion
• Microinjections
• LASER

6. IN-DIRECTMETHODOF
TRANSFORMATION
 Agrobacterium tumefaciens
02

7. Classification
 Kingdom: Bacteria
 Phylum: Proteobacteria
 Class: Alproteobacthaperia
 Order: Rhizobiales
 Family: Rhizobiaceae
 Genus: Agrobacterium
 Species: A. tumefaciens

8. AgrobacteriumgeneralIntroduction
 Rod shape
 Gram negative
 Found in soil
 Dicot infection
 Cause diseases like Crown gall and hair roots in dicot plants
 The bacteria transfers a tumor-inducing plasmid

9. • Hairy root disease
• Root-inducing plasmid (Ri)
01
02
• Cause crown-gall disease
• T-DNA
• Tumor-inducing plasmid (Ti)
Agrobacterium tumefaciens
Agrobacterium rhizogenes
TypesofAgrobacterium

10. Ti-Plasmid
 Double strand
 Circular
 conjugative
 Part of Ti-plasmid that cause tumor is T-DNA
 Size: 150-250kb
 Ti-plasmid disappear above 28°c temperature
 Two regions necessary for transmission is Virulence region and T-DNA

11. Cont…..
Ti-plasmid classified according to the Opines produce
 Nopaline plasmids:
 Carry gene for synthesizing nopaline in the plant and for utilization in bacteria.
 Tumors can differentiate into shoot masses.
 Octopine plasmids:
 Carry gene for synthesizing octopine in the plant
 Tumor do not differentiates, but remain as callus tissue

12. PartsofTi-Plasmid
VirulenceRegion
• Needed for
T-DNA
transfer
OpineCatabolism
• Required for a
breakdown of
Opine
OriginofReplication(ORI)
• Start the replication
of Ti-plasmid
T-DNA Region
• Tumor induction
• Produces plant hormone
• It consists of : Auxin, Cytokinin, and Opines
• Transferable part
• Oncogenic region

13. T-DNA
 The T-DNA region of is defined by the presence of right and left border sequence in Ti-plasmid
 The border are 25bp of directly repeated sequences of T-DNA
 One or both border region provide the side for the recognition of gene products from the vir region.
LB
auxA auxB cyt ocs
RB
LB, RB: Left border and Right border
auxA, auxB: Enzyme that produce auxin
Cyt: Enzyme that produce cytokinin
Ocs: Octopine synthase, produce
octopine
Fig. T-DNA

14.  Genes in the vir region play major role in T-DNA transmission
 Acetosyringone (AS) a flavonoid is released by wounded plant cells activates vir genes
 Vir A, B, C, D, E, F, G : 7 complementation groups, but some have multiple ORFs
 30kb of Ti-plasmid
Fig: The composition of the vir region of octopine-type Ti plasmids
Virulenceregion

15. Vir genesandtheirfunction
• Sense acetosyringone
secreted by wounded
plant cell
Function
Vir A Vir G
• Transcriptional activator
of Vir box
Function
• Produce endonuclease
• Cut T-DNA at right border
to initiate T-strand
synthesis
• Protects 5’ end from being
cleaved by exonuclease
Function
• Helps vir D2 to recognize
and cleave within the
25bp border sequence
Function
Vir D2 Vir D1

16. Vir genesandtheirfunction
• Forms overdrive
sequence(cis-active 24
bp sequence adjacent to
the right border)
• Helps in DNA transfer
Function
Vir C Vir E2
• Binds to T-strand protecting
from nuclease attack
• It introduce with lipids to
forms channels in the plant
membranes through which
the T-complex passes
Function
• Act as a chaperone of vir E2
which help in stabilization in
Agrobacterium
Function
• Forms conjugational pore
between plant and
bacteria
Function
Vir E1 Vir B

17. Chromosomal VirulenceGene
• Major Role in
exopolysaccharide
production
chv A and B
• Major role in T-DNA
transport
psc A
• Glucose and Galactose
transport
chv E
• Virulence property
Chv D, ilv. miaA and att

18. AGROBACTERIUM
MEDIATED GENE
TRANSFER
03

20. Somefacts
 The continued
presence of
viable bacteria
is not needed
for tumor
maintanance
 Bacteria do not
penetrate into plant cell
that are converted into
tumor cells
 Only small part of the
Ti-plasmid is
trasnferred into the
host cell. This segment
is called as T-DNA
1
2
3

21. THE PROCESS OF
T-DNA TRANSFER
AND
INTEGRATION
04

22. Theprocess ofT-DNAtransferandintegration
02
01 03 04 06
05
Signal
recognition by
agro bacterium
Attachment to
plant cells
Induction of vir
genes
T-Strand
production
Transfer of T-DNA
out of the
bacterial cell
Transfer of the T-
DNA into the plant
cell and nuclear
localization

23. Steps
Steps 1: Signal recognition by agrobacterium
 Recognition of the site is done by the Acetosyringone Compound
 This compound is produced by the wounded plant cell

24. Step 2. Attachment of Plant cells
 Two Steps:
 Initial attachment via Polysaccharide
 Mesh of cellulose fiber produced by bacteria
 Chromosomal virulence genes are involved in the attachment
 Chv genes

25. Step 3. Induction of vir genes
 virA gene sense acetosyringone secreted by the plant
 virA gene activates virG
 virG induces expression of all the vir genes
 Chromosomal vir gene chvE encodes glucose and galactose which enhance the vir gene induction

26. Step 4. T-strand production
 VirD1- VirD2 complex
 It recognized the Right border and Left border
 It encodes a site-specific cutting enzyme
 It cuts T-DNA borders at 3rd and 4th bases
 VirD2 attaches to the 5’ end of T-DNA and protects 5’end cleaved by exonuclease
 VirC1
 It assists in this process
 It recruits the T-DNA complex at the pole to interact with virD4

27. Step 5. Transfer of T-DNA out of the bacterial cell
 For delivery of T-DNA from bacteria to plant plasma membrane require a Type IV secretion system
 Type IV secretion system/ T4SS
 Also called T4SS
 It is a secretion protein complex found in bacteria
 It able to transport protein and DNA across the cell membrane
 Related to conjugation
 Contain 12 proteins (virB1-virB11 and virD4)
 Protein forms 2 functional components
i. Ti pilus
ii. Membrane-associated transporter complex

28. Cont..
 Transport complex
 Cylindrical structure
 Consist of inner and outer membrane
 virD2: Act as a substrate receptor
 VirB3, B4, B6, B8, and B11: Inner
membrane translocase
 virB7, B9, B10: Outer membrane core
complex
 virB2, virB5: extra cellular Ti pilus

29. 6. Transfer of the T-DNA into the plant cell and nuclear localization
 T-complex
 It contains T-DNA, virD2, and virE2
 virD2
 Protect the 5’ end of T-DNA
 It consists of nuclear localization signal which facilitates its interaction with plant protein
 virE2
 It coats the T-strand and protects from degradation
 It interacts with VIP2 (nuclear factor) to mediate interaction with chromatin and facilitates
integration

30. Contd…
 Inside the nuclease, ss-TDNA is converted into ds-TDNA which gets integrated into the
plant genome via a process called Illegitimate recombination
 Illegitimate recombination: The process by which two unrelated double-stranded
segments of DNA are joined

32. Advantages
 This is the normal method of gene transfer
 It can transform a wide range of dicotyledons
plants
 Large-size DNA can be transferred
 Stability of transferred DNA is high
 Transformed plant can be regenerated
effectively
Disadvantages
 Ti-Plasmid are large size, smaller vectors are
preferred in rDNA technology
 Absence of unique restriction enzyme
 Only a few monocots are transformed
 It release phytohormones so may cause
crown gall unknowingly

33. Diseasecausedbyagrobacterium

34. THNAKYOU