agrobacterium

•Introduction
•Biology of Agrobacterium
•Ti plasmids—
Introduction
Organization
Types
•T-DNA transfer and integration
•Ti vectors
Co-integrate vector system
Binary vector system
•Agrobacterium mediated transformation
Leaf disk transformation
In planta transformation
Application and limitations
• Conclusion
• References

INTRODUCTIO
N
Ti stands for tumour inducing plasmid.
Plant genetic transformation technique basically deals with the
transfer of desirable gene from one plant species to another with
subsequent genome integration and expression of the foreign gene
in host.
The plants obtained through genetic engineering contain a gene or
genes usually from an unrelated organism; such genes are called
transgenes, and the plant containing transgenes are known as
transgenic plants.

Biology of Agrobacterium
Agrobacterium tumefaciens is a rod shaped, gram negative, soil born motile
bacterium that uses horizontal gene transfer to cause tumor in plants. This
bacterium infects the parts of the plant which are in contact with soil.
The genus includes four species of bacteria. They are A. tumefaciens, A.
radiobactor, A.rubi and A. rhizogenes.
invades many dicotyledonous plants and some gymnosperms when
they are damaged at soil level.
enters the fresh wound and attaches itself to the wall of an infected
cell, after which it transfers some of its DNA into the chromosome of
its host plant cell.

According to Zaenen et al. (1974) and Schell (1974) almost all the
strains of A. tumefaciens contain Ti plasmid.
Fraley et al. (1983) exploited the natural ability of A. tumefaciens to
transfer DNA into plant genome.
Some workers consider this bacterium as the natural expert of
interkingdom gene transfer. The major credit for the development of
plant transformation technique goes to the natural unique capability of
Agrobacterium tumefaciens thus this bacterium is the most beloved by
plant biotechnologists.
There are mainly 2 species of Agrobacterium-
1) Agrobacterium tumefaciens that causes crowngall disease.
2) Agrobacterium rhizogenes that causes hairy root disease.

Biology of Agrobacterium
Fig: - Agrobacterium cells Ref:- www.plantvector.com

a bacterium was the
causative agent of
crowngall tumour although
its importance was
recognized much later. Smith and
Townsend
These cells are capable of growing on a growth regulator (GR) - free
medium, while normal plant cells need exogenous auxin and cytokinin.
They also synthesize unique nitrogenous compound called opines,
which are neither produced by normal plant cells nor utilized by them.
Agrobacterium cells use opines as their carbon and nitrogen source; the
bacteria are usually present in the intercellular space of crowngalls.
Properties of crowngall cells

Properties of crowngall cells
Fig: - plant showing crown gall disease Ref: - http://4e.plantphys.net/article

The Ti plasmid
Size-- about 200kb (range 150-250kb).
pTi is lost when grown above 28C,. they become avirulent.
Weight-- range from 120-160 mega Daltons.
The plasmid has 196 genes that code for 195 proteins. The GC content is 56% and
81% of the material is coding genes.
The pTi are unique bacterial plasmid in the following 2 respect—
•They contain some gene (the gene located in their T-DNA), which have regulatory
sequence recognized by plant cells, while their remaining genes have prokaryotic
regulatory sequence.

The Ti plasmid
Fig: - the Ti plasmid Ref: - www.Ti-plasmid.com

The Ti plasmids have 5 important regions—
1)T-DNA region
2)vir region
3)Opine catabolism
region
4)tra region
5)Ori region
the bio-synthesis of auxins, cytokine and
opine and is flanked by left and right borders.
Size-- 23-25kb long fragment
eukaryotic regulatory sequence to produce
growth hormones like auxin and cytokinin.
Therefore, they express themselves in plant
cells only and produce crown gall in plants.
Flanked by 24 bp direct repeat border
contains genes that code for enzymes
synthesizing opines By transferring the T-DNA
into the plant genom.

1)T-DNA region
2)vir region
3)Opine catabolism
region
4)tra region
5)Ori region
T-DNA is composed of following two regions—
a) Onc-region
b) Ops-region
Onc- regions: 3 types of genes- tms-1 and tms-2
genes for shooty locus, tmr-gene for rooty locus.
They produce IAA, IPA-5mp. Here IAA is an auxin and
IPA-5mp is a cytokinin. These three genes code the
enzymes responsible for the synthesis of these
phytohormones. Thus, if these three genes are
inserted into nuclear genome of plant then plant
becomes able to produce phytohormones.
Ops-regions: synthesis of unusual amino acids or
sugar derivatives called as Opines. Opines are
formed of arginine and pyruvate like compounds
found in the cells.

1)T-DNA region
2)vir region
3)Opine catabolism
region
4)tra region
5)Ori region
Size-- 40 kb of DNA and have 25 genes.
contains 8 operons (designated as virA, virB,
virC, virD, virE, virF, virG and virH),
induce the transfer of T-DNA, hence essential
for virulence.
4 operons, viz., virA, virB, virD, and virG, are
essential for virulence.
virA codes for a receptor which reacts to the
presence of phenolic compounds such as
acetosyringone which leaks out of damaged
plant tissues.

1)T-DNA region
2)vir region
3)Opine catabolism
region
4)tra region
5)Ori region
This regions code for
protein involved in
the uptake and
metabolism of
opines.

1)T-DNA region
2)vir region
3)Opine catabolism
region
4)tra region
5)Ori region
Conjugative transfer
region functions in
conjugative transfer
of the plasmid; it can
also function in T-
DNA transfer when
the T-DNA borders or
deleted.

1)T-DNA region
2)vir region
3)Opine catabolism
region
4)tra region
5)Ori region
This region is
responsible for origin
of replication for
propagation in
Agrobacterium

Types of Ti plasmid
Opines are low molecular weight compounds found in
plant crown gall tumors.
Opine biosynthesis is catalyzed by specific enzymes
encoded by T-DNA which is part of the Ti plasmid,
inserted by the bacterium into the plant genome.
The opines are used by the bacterium as an important
source of nitrogen and energy.
Each strain of Agrobacterium induces and catabolizes a
specific set of opines. There are at least 30 different
opines described so far.

The very first opine discovered, octopine, was initially isolated from octopus
muscle.
Similar derivatives have been isolated from muscle tissue of certain marine
invertebrates.
Opines like acetopine and nopaline can also be formed in normal callus and
plant tissue as a result of arginine metabolism.
i) octapine Ti plasmids:
associated with the catabolism of octapine Octapine is an unusual amino acid
synthesized from arginine. The molecular formula is C9H18N4O4. E.g.., pTiB6,
pTiAch5, pTiB653.
ii)Nopaline Ti plasmids:
associated with the catabolism of Nopaline. The molecular formula is C19H16N4O6.
e.g.-- The pTiC58 is 194000bp in size.
iii) Leucinopine Ti plasmids:
iv) Succinamopine Ti plasmid:
classified following four kinds

Types of Ti plasmid
Fig: - the Ti plasmid Ref: - www.Ti-plasmid.com

T-DNA transfer and integration
1) Signal induction to
Agrobacterium
2) Attachment of Agrobacterium to
plant cells
3) Production of virulence protein
4) Production of T-DNA transfer
5) Transfer of T-DNA out of
Agrobacterium
6) Transfer of T-DNA into plant cells
7) Integration of T-DNA
The wounded plant cells release
certain chemicals phenolic
compound such as
acetosyringone and
hydroxyacetosyringone and
sugars which are recognized as
signals by Agrobacterium.
The signals induced result in a
sequence of biochemical events in
Agrobacterium that ultimately
help in the transfer of T-DNA.

1) Signal induction to
Agrobacterium
plant cells
Agrobacterium
6) Transfer of T-DNA into plant
cells
They have flagella that allows them to swim through
the soil towards plants and accumulate in the
rhizosaphere around roots.
an initial weak and reversible attachment
they synthesize cellulose fibers that anchor them to
the wounded plant cells.
Several chromosomal virulence (chv) genes
responsible for the attachment of bacterial cell to
plant cells have been identified.
Four main genes are involved in this process are:
chvA, chvB, pscA and att. It appears that the
products of the first three genes are involved in the
actual synthesis of the cellulose fibrils. These fibrils
also anchor the bacteria to each other, helping to
form a micro-colony.

1) Signal induction to Agrobacterium
plant cells
Agrobacterium
after attachment a series of events take place
that result in the production of virulence
protein.
The virA/virG two component sensor systems
is able to detect phenolic signals released by
wounded plant cells.
This leads to a signal transduction event
activating the expression of 11 genes within
the virB operon which are responsible for the
formation of T pilus.
Thus the signal phenolic molecules bind to
VirA protein (associated with ChvE proteins),
which then phosphorylates itself and VirG
protein, a DNA binding protein.
Phosphorylated VirG induces the transcription
of all the 8 vir operons.

plant cells
Agrobacterium
The phenolic signal molecules bind to the
virA gene product, the VirA protein. VirA
functions in conjugation with the protein
ChvE (a sugar binding protein).
The activated VirA functions as an autokinase
and phosphorylates itself. The
phosphorylated VirA then phosphorylates
VirG protein, which then, most likely
dimerises and induces the expression of all
the 8 vir operons.
Thus the signal phenolic molecules bind to
VirA protein (associated with ChvE proteins),
which then phosphorylates itself and VirG
protein, a DNA binding protein.
Phosphorylated VirG induces the
transcription of all the 8 vir operons.

plant cells
Agrobacterium
The right and left borders are recognized by
virD1/virD2 proteins involved in the
production of single standed T-DNA.
The virD1 gene product, virD1 protein, has
topoisomerase activity; it binds to the right
border sequence, and relaxes supercoiling,
which facilitates the action of protein VirD2.
VirD2 is an endonuclease; it nicks at the right
border and covalently binds (and remains
bound during the T-DNA transfer) to the 5’-
end so generated. The T-strand is again nicked
at the left border to generate a single-strand
copy of T-DNA.
VirE2 protein is a single-strand binding
protein; about 600 copies of it bind to the
single-strand T-DNA and protect it from
nuclease action.

plant cells
Agrobacterium
The ssT-DNA – VirD2 complex in association
with virG is exported from the bacterial cells.
VirB products froms the transport apparatus.
The virB operon has 11 genes, which encode
mostly membrane-bound proteins.
Most likely VirB proteins, together with
VirD4 protein, participate in conjugal tube
formation between the bacterial and plant
cells, which provides a channel for T-DNA
into the plant cell.
1st the virB pro-pilin is formed. This is a
polypeptide of 121 amino acids which
requires processing by the removal of 47
residues to form a T pilus subunit. The
subunit is circularized by the form of a
peptide bond between the two ends of the
polypeptide.

plant cells
Agrobacterium
the endonuclase VirD2, which nicks the right
border and remains bound to the 5’-end of the
single-strand T-DNA copy, has a signal
sequence, which drives it towards the nucleus
of the transformed plant cell.
The T-DNA virD2 complex cross the plant
plasma membrane. In the plant cells, T DNA
gets covered with the virE2. This covering
protects the T-DNA from degradation by
nucleases virD2 and virE2 interact with a
variety of plant protein which influence T-DNA
transport and integration.
The T-DNA virD2, virE2-plant protein complex
enters the nucleus through nuclear pore.
Nuclear localization signals or NTS, located on
the virE2 and virD2 are recognized by the
importin.

plant cells
Agrobacterium
T-DNA enters plant cells in a single-
standed form; it is immediately
converted into a double-standed form in
the nuclei.
It has been suggested that the high
frequency of transformation by
Agrobacterium is due to the single-
stranded T-DNA transfer.
The double-stranded T-DNA integrates
at random sites in the host plant
genome most likely by a process of
recombination due to a homology in
short segments of the host DNA.
The T-DNA is generally integrated in low
copy numbers per cell, but up to1 dozen
copies/cell have been recorded.

plant cells
Agrobacterium
Most of genes that are located
with in the T-DNA region are
activated only after the T-DNA is
inserted into the plant genome.
The products of these genes are
responsible for crown gall
formation. Both auxin and the
cytokinins regulate plant cell
growth and development.
In excess, they can cause the plant
to develop tumorous growths such
as crown galls.

Fig: - transfer of T-DNA Ref: - www.Ti-plasmid.com

Crown gall formation

Ti plasmid derived vector system
Agrobacterium-mediated T-DNA transfer is widely used as a tool in
biotechnology.
In genetic engineering, the tumor-promoting and opine-synthesis
genes are removed from the T-DNA and replaced with a gene of
interest and/or a selection marker.
Agrobacterium is then used as a vector to transfer the engineered T-
DNA into the plant cells where it integrates into the plant genome. This
method can be used to generate transgenic plants carrying a foreign
gene.

The use of wild type pTi as a vector presents the following problems—
1) Presence of oncogenes in T-DNA.
2) The Ti plasmid is too large to manipulate easily in vitro.
3) A general lack of unique cloning sites with in the T-DNA.
4) Opine production in transformed plant cell lowers the plant yield.
5) Ti plasmids cannot replicate in E. coli this limits their utility as E. coli.
These problems have been solved by-
1) Deleting the oncorenes from the T-DNA (Disarming).
2)Two strategies evolved to overcome this manipulation difficulty.
Both strategies-- hormone and opine synthesis genes in are not needed.
The vir genes, essential for transfer of T-DNA, are located outside of T-DNA
on the Ti plasmid.
One of the introduced genes is usually an antibiotic resistance gene
allowing selection of transformed plant cells on antibiotic-containing media.

Fig: - Ti plasmid derived vector Ref : - www.phytovector.com

Co-integrate vector system:-
In the co-integrate vector system, the disarmed and modified Ti plasmid
combines with an intermediate cloning vector like E. coli plasmid to produce a
recombinant Ti plasmid.
The co-integration of the two plasmids is achieved within Agrobacterium by
homologous recombination. Therefore, the E.coli plasmid, e.g. - pBR322, and
the disarmed pTi must have some sequence common to both for recombination
to occur.
Production of Disarmed Ti plasmid: - the deletion of genes governing hormone
biosynthesis T-DNA of a Ti plasmid is known as disarming. In place of deleted
DNA a bacterial plasmid pBR322 DNA sequence is incorporated. This disarmed
plasmid, has the basic structure of T-DNA necessary to transfer the plant cell.
However the cells containing the modified T-DNA were nontumorous.

Construction of intermediate vector: -
The intermediate vector is constructed with the following component—
1) A pBR322 sequence DNA homologous to that found in the receptor Ti
plasmid.
2) A plant transformation marker e. g. – a gene coding for neomycin ,
phosphotransferase II.
3) A bacterial resistant marker e. g. - a gene coding for spectinomycin
resistance. This gene confers spectinomycin resistant to recipient bacterial
cell and thus permits their selective isolation.
4) A MCS region where foreign gene can be inserted.
5) A origin of replication which allows the replication of plasmid in E. coli.
But not in Agrobacterium.

The vir region of Ti plasmid need not be present in the same plasmid for an
efficient transfer of T-DNA.
The T-DNA present in one plasmid (which does not have the vir region) is
readily transferred into plant cells in response to vir genes present in another
plasmid contained in the same bacterial cell. This property has been exploited
to construct binary vectors of pTi.
The binary vector system consists of an Agrobacterium strain along with a
disarmed Ti plasmid called vir helper plasmid (the entire T-DNA region including
borders deleted while Vir gene is retained). It may be noted that both of them
are not physically linked.

Production and use of binary vector:-
A binary vector consist of a pair of plasmids of which one plasmid contains
disarmed T-DNA sequence (the right and left border must present), while the
other contains the vir region, and ordinarily lacks the entire T-DNA including the
border.
The plasmid containing the disarmed T-DNA is called mini-Ti, and has the
origins for both the E. coli and Agrobacterium. The DNA insert is integrated
within the T-region and the recombinant miniTi is cloned in E. coli.
Mini-Ti has kan- kanamycin resistance gene for the selection of Agrobacterium
cells and neo- gene for the selection of transformed plant cells.
The helper plasmid is a Ti plasmid having a functional vir region but lacking the
T-DNA region, including the border sequence. The transformed plant cells can
be selected on kanamycin medium due to the neo gene present with in the T-
DNA.

Fig: - Binary vector system Ref : - www.phytovector.com

Fig: - Co-integrate vector system Ref : - www.phytovector.com

Agrobacterium mediated transformation
Transformation with Agrobacterium can be achieved in two ways:-
1) Coculture with tissues explant: -
the recombinant T-DNA is placed in Agrobacterium, which is then
cocultured with the plant cell or tissue to be transformed for about 2 days.
In case of many plants small leaf disks are excised from surface- sterilized
leaves and used for cocultivation e.g.- tomato, tobacco.
During this culture acetosyningone released by plant cells induces the vir gene,
which together brings about the transfer of T-DNA into many of the plant cells.
Now when this leaf disks are allow to grow in medium containing kanamycin
only transformed cells are allow to grow. They divide and regenerates shoots
within 3-4 weeks.

1) Coculture with tissues explant: -

2) In Planta Transformation: -
A common transformation protocol for Arabidopsis is the floral-dip method:
the flowers are dipped in an Agrobacterium culture, and the bacterium
transforms the germline cells that make the female gametes.
The seeds can then be screened for antibiotic resistance (or another marker
of interest), and plants that have not integrated the plasmid DNA will die. It
appears that Agrobacterium cells enter the seedlings during germination,
and retained with in the plant, and when flowers develop they transform the
zygote.
Agrobacterium is listed as being the original source of genetic material that
was transferred to these USA GMO foods. E.g.- Soybean, Cotton, Corn, Sugar
Beet, Alfalfa, Wheat and Rice (Golden Rice)
Economically, A. tumefaciens is a serious pathogen of grape vines, , nut trees,
and sugar beets.

Advantage of Agrobacterium mediated transformation
•This is a natural method of gene transfer.
•Agrobacterium can conveniently infect any explant (cell/tissue).
•Even large fragment of DNA can be effectively transferred.
•Stability of transferred DNA is reasonably good.
•Transformed plant can be regenerated effectively.
Limitations
•it is a costly process.
•There is a limitation of host plants for Agrobacterium, since many crop plants
(monocotyledons) cereals are not infected by it.
•The cells that regenerate more efficiently are often difficult to transform. E. g.
embryonic cells.

References: -
S. No. Book Author Edition
1 Molecular biotechnology Pasterneck 2005
2 Molecular biotechnology Channarayappa 2006
3 Introduction to applied
biology and biotechnology
k. Vaidyanath 2006
4 Biotechnology U. Satyanarayan Reprint 2009
5 Biotechnology– expanding
horizon
B. D. Singh 2007
search engines: -
http://4e.plantphys.net/article
www.Ti-plasmid.com
search engines: -
http://4e.plantphys.net/article
www.Ti-plasmid.com

agrobacterium

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to agrobacterium

Similar to agrobacterium (20)

More from Akanksha Golchha

More from Akanksha Golchha (13)

Recently uploaded

Recently uploaded (20)

agrobacterium