Methods of gene transfer, about agrobacterium, about Ti-plasmid, about Ri-plasmid, preparation of vectors - co-integrated and binary, gene transfer process.

1. Shovan Das
B.Sc (Agriculture)
Bidhan Chandra Krishi Viswavidyalaya
AGROBACTERIUM MEDIATED
GENE TRANSFER

2. 1
Transferring of gene is necessary to incorporate foreign genes or modified genes (of that
organism) into an organism. This process is called transformation and the produced
individual is called transgenic. There are various methods of gene transfer, agrobacterium
mediated transfer is one among them.
Methods of gene transfer:
 Direct transfer: Directly transfer gene into an organism.
1. Physical methods: Micro injection, macro injection, pressure, biolistic-gene
gun/particle bombardment, electroporation, silicon carbide fibres, laser
mediated, liposome encapsulation,
2. Chemical methods: Polyethylene glycol (PEG) method, diethyl amino ethyl
(DEAE) dextran mediated, calcium phosphate precipitation, artificial lipids,
proteins, dendrimers.
 Indirect transfer:
1. Biological methods: Agrobacterium tumifaciens mediated, Agrobacterium
rhizogenes mediated, plant viruses.
 In plants: Meristem transformations, floral dip method, pollen transformation.
So, agrobacterium mediated gene transfer is a biological method where gene is inserted
indirectly by using a vector.
Why agrobacterium?
a. It successfully infects the host and transfers
the target gene into it, high transformation
efficiency.
b. Simple and comparatively less expensive.
c. Large number of dicots and few monocots
(as they don’t produce phenolic) and
gymnosperms response to agrobacterium.
d. Transgenic crops obtained have better fertility percentage.
About agrobacterium:
 Discovered by Smith and Townsend (1907).
 A soil-born gram negative bacterium causing induction of ‘crown gall' and ‘hairy root'
diseases. It is rod shaped and motile, having 1-6 peritichous flagella.
 It is a phyto-pathogen, and it is regarded as Nature’s most effective plant genetic
engineer. It is the natural expert of inter-kingdom gene transfer.
 There are three strains of agrobacterium –
Strains Causes
Agrobacterium tumefaciens Crown gall disease
Agrobacterium rhizogenes Hairy root disease
Agrobacterium radiobacter Avirulent strain
Agrobacterium Mediated Gene Transfer
Crown gall in sugar beet caused by
agrobacterium

3. 2
Agrobacterium contains a chromosome & a
plasmid. Both chromosomes & plasmid
contains different genes which help in the
transfer of the foreign DNA into host.
The plasmid is Ti (tumour inducing) plasmid or
Ri (root inducing) plasmid. Only the T-DNA
part of plasmid is inserted into the host cell,
where the foreign gene recombined.
Chromosomal genes:
Chromosomal genes Function/s
Chv A& B Major role in exopolysaccharide production.
psc A T-DNA transport.
chv E Glucose and galactose transport.
chvD, ilv, miaA and att Have virulence property.
Ti Plasmid:
Ti plasmid is a large conjugative plasmid or mega plasmid of about 200 kb. Ti plasmid is a
circular vector containing various regions
which have different functions –
 T-DNA region: T-DNA is 23 kb long
containing various genes which have
regulatory sequences recognised by
plant cells. It initiates at right border
(RB) and terminates at left border
(LB). These are not transferred intact
to the plant genome, but are involved
in the transfer process. The RB is
rather precise, but the LB can vary by
about 100 nucleotides. Deletion of the
RB repeat abolishes T-DNA transfer,
but the LB seems to be nonessential. At least RB should present in plasmid for
successful transformation. This region has genes for auxin, cytokinin & opine
synthesis. Auxin & cytokinin are useful for plant (they stimulate the growth of that
definite plant part, which results the formation of tumour or crown gall). But opines
are not useful to plants, rather it is used by the bacterium for C & N source. Genes
present in T-DNA and their functions are –
Gene Product Function
ocs Octopine synthase Opine synthesis
nos Nopaline synthase Opine synthesis
trns1 (iaaH, auxA) Tryptophan-2-mono-oxygenase Auxin synthesis

4. 3
trns2 (iaaM, auxB) Indoleacetamide hydrolase Auxin synthesis
trnr (ipt, cyt) Isopentyltransferase Cytokinin synthesis
trnL Unknown Unknown, mutations affect
tumour size
frs Fructopine synthase Opine synthesis
mas Mannopine synthase Opine synthesis
ags Agropine synthase Opine synthesis
 Opine catabolism region: Genes are carried on this region allow the bacterium to
utilize opines as nutrient.
 Origin of replication (Ori): Replication of DNA of that plasmid starts from here.
 Virulence region: Virulence region (30 kb) consists of 24 genes which code for
proteins that prepare the T-DNA and the bacterium for transfer. Virulence genes are
located in 8 operons from virA to virH. vir A,F,G are monocistronic operons, whereas
vir B,C,D,E,H are polycistronic.
Operons Function/s
virA Chemoreceptor (senses acitosyringone & alpha hydroxy acitosyringone),
activator of virG. It is activated after binding with phenolic compound and
cause auto-phosphorylation due to auto-kinase activity on histidine residue
of vir A.
virB Transmembrane complex (conjugational pores between plant cell and
bacteria). VirB11 has ATPase activity and generate ATP needed for the
delivery of T-DNA into the plant cells.
virC Host-range specificity. Helps in DNA transfer (VirC1 specifically binds to
overdrive sequence and stimulates the transfer process. Agrobacterium
tumefaciens uses type IV secretion system [T4SS] to transfer T-DNA
complex to its host cells. T4SS also known as mating pair formation
apparatus is a cell envelope spanning complex. T4SS form a pore or
channel).
virD Site-specific endonuclease, essential for cleavage of super coiled stranded
substrate. VirD1 has topoisomerase activity and VirD2 has endonuclease
activity.
virE T-DNA processing and protection (it bind to the single stranded DNA and
protect it from nuclease action).
virF Host range specificity. It directs the T-complex (virD4, virB1, virB2, T4SS)
protein for destruction in proteosomes.
virG Positive regulator of vir B, C, D, E, F. (virA cause phosphorylation to VirG
protein and bind with virG forming dimer and then induce the expression of
other operons.
virH Encodes P-450 type monooxygenases protein. Associated with
detoxification of a variety of compound.
 Agrobacterium tumeficiens strains generally produce octapine (arginine + alanine) or
nopaline (arginine + glutamine).
 Agrobacterium rhizogenes produce either agropine or mannopine.

5. 4
Ri Plasmid:
The Ri–plasmid contains a distinct segment of DNA which is transferred to plant genome
during infection. The A. rhizogenes have Ri plasmid. Strains of A. rhizogenes are known to
produce agrocinopine and few opines of the agropine group.
Preparation of vectors:
The preparation of vectors is essential for transferring the foreign DNA (target DNA/gene of
interest) to the host (plant). There are two kinds of vectors are used for transfer –
1. Co-integrated Vectors: This is the type of vector where T-DNA region and virulence
region are co-integrated. Three vectors are required in this system –
Vectors /
Plasmids
Initially
resides in
Features & functions
Disarmed Ti
plasmid
A. tumifaciens Oncogenes (phytohormone genes which produce auxin &
cytokinin, as they are responsible for tumour formation)
located in T-DNA region are replaced by exogenous DNA,
so it is disarmed. They are pGV3850 vector with left &
right border of T-DNA region.
Intermediate
vectors
E. coli (then
transferred to
agrobacterium
through
conjugation)
These are small pBR322 based plasmids containing a T-
DNA region with foreign DNS included. They are
replicated in E.coli and are transferred into agrobacterium
by conjugation. They have only origin pf replication for E.
coli (OriE), so they can’t replicate in agrobacterium. They
also need DNA segments homologous to the disarmed T-
DNA to permit recombination to form a co-integrated T-
DNA structure.
Helper
vectors
E. coli (stays
here, are not
transferred).
These are small plasmids contain transfer (tra) and
mobilization (mob) genes, which allow the transfer of the
intermediate vectors into Agrobacterium.
A resulting co-integrated plasmid assembled by in vitro manipulation normally contains: the
vir genes, the left and right
T-DNA borders, an
exogenous DNA sequence
between the two T-DNA
borders, and plant and
bacterial selectable markers.
These vectors were among
the first types of modified
and engineered Ti plasmids.
There are several drawbacks
of using co-integrated
vectors, so that they are not
widely used today.

6. 5
 Drawbacks of co-integrated vectors:
1. Long homologies required between the Ti plasmid and the E. coli plasmids
making them difficult to engineer and use.
2. Relatively inefficient gene transfer compared to the binary vectors.
2. Binary Vectors: 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 (the vir genes) and the disarmed T-DNA containing the transgene on separate
plasmids. The two different plasmids employed are –
Vectors / Plasmids Initially
resides in
Feature & functions
Small replicon E. coli
(transferred to
agrobacterium)
It has an broad origin of replication (RK2) that
permits the maintenance of the plasmid in a wide
range of bacteria including E. coli and
Agrobacterium. This plasmid typically contains:
– Foreign DNA in place of T-DNA,
– The left and right T-DNA borders (or at least the
right T-border),
– Markers for selection and maintenance in both E.
coli and A. tumefaciens,
– A selectable marker for plants.
Ti plasmid (with vir
region)
A. tumifaciens It lacks the entire T-DNA region but contains an
intact vir region.
 The T-DNA plasmid can be introduced into Agrobacterium by triparental mating or
by a more simple transformation procedure, such as electroporation.
 These two plasmids resides separately in agrobacterium, they don’t need
recombination. So here the adaptability is wide.
 The plant cells are co-cultivated with the Agrobacterium, to allow transfer of
recombinant T-DNA into the plant genome and transformed plant cells are selected
under appropriate conditions.
Advantages of binary vectors:
 Small size due to the absence of border sequences needed to define T-DNA region
and vir region.
 Ease of manipulation.
Examples of binary vectors:
 pBIN19: one of the first binary vectors developed in 1980s and was widely used.
 pGreen: A newly developed vector with advanced features than pBIN19.

7. 6
T-DNA transfer and integration:
 Signal recognition by Agrobacterium: The
wounded plant cells release certain chemicals,
such as phenolic compounds and sugars. These
chemicals are recognized by Agrobacterium as
signals. This in turn results in a sequence of
biochemical events in Agrobacterium that helps in
transfer of T-DNA of Ti plasmid.
 Attachment to plant cell: Attachment of this
bacterium to plant cells is a two-step process. It
involves an initial attachment via polysaccharides
(the product of att R locus). Subsequently, a mesh of
cellulose fibres is produced by Agrobacterium.
Several chromosomal virulence genes (chv genes)
are involved in attachment of bacterial cells to the
plant cells.
 Induction of virulence gene: virA (a membrane-
linked sensor kinase) senses phenolic compounds
(such as acetosyringone) and autophosphorylates,
subsequently phosphorylating and, thereby,
activating virG. This activated virG induces
expression of other virulence gene of Ti plasmid to
produce the corresponding virulence proteins (D, D2,
E2, B). It has been also identified that certain sugars
(e.g. glucose, galactose, xylose etc.) also induce
virulence gene.

8. 7
 Production of T-DNA
strand: The right and left
border sequence of T-
DNA are identified by
virD1/ virD2 protein
complex and virD2
produces single stranded
DNA (ss-T-DNA). After
nicking, virD2 becomes
covalently attached to the
5'end of ss-T- DNA strand
and protect and export the
ss-T-DNA to plant cells.
 Transfer of T-DNA out
the bacterial cell: The ss-
T-DNA – virD2 complex
in association with virE2 is exported from bacterial cell by a ‘T-pilus' (a membrane
channel secretary system).
 Transfer T-DNA into plant cell and integration: The single stranded T-DNA–
virD2 complex and other vir proteins cross the plant plasma membrane. In the plant
cells, T-DNA gets covered with vir E2. This covering of virE2 helps in protection of
ss-T-DNA from degradation by nucleases. virD2 and virE2 interact with variety of
plant proteins which influence the T-DNA transport and integration. The T-DNA –
virD2 – virE2 – plant proteins complex enters the nucleus through nuclear pore
complex (NPC). In the nucleus, T-DNA gets integrated into the plant genome by a
process referred to as ‘illegitimate recombination'. This process is unlike homologous
recombination as it does not depend on extensive region of sequence similarity.