BY:

DR. JAMILAH SYAFAWATI BINTI YAACOB, PhD

(Tel: 03-
03-79674090, Fax: 03-
03-79674178)

INSTITUTE OF BIOLOGICAL SCIENCES,

FACULTY OF SCIENCE,
UNIVERSITY OF MALAYA,
50603 KUALA LUMPUR,
MALAYSIA.
TRANSFORMATION APPROACHES

(AGROBACTERIUM
AGROBACTERIUM-
AGROBACTERIUM-MEDIATED
TRANSFORMATION & DIRECT
TRANSFORMATION)
Reading Materials

GENETICALLY MODIFIED
ORGANISMS (2003)
Yves tourtes
PLANT BIOTECHNOLOGY
(2003) - Adrian Slater, Nigel
Scott, Mark Fowler
Agrobacterium tumefaciens:
a natural tool for plant
transformation, Electronic
journal of biotechnology: Vol
1, No3, Dec 15, 1998
Essential requirements for plant genetic
engineering:

a) Suitable tissue culture system

b) Methods of gene transfer into plants
c) Suitable gene/construct
d) Gene analysis
e) Field testing
Must get DNA:
1. into the cells
2. integrated into the genome (unless using transient
expression assays)
3. expressed (everywhere or controlled)

For (1) and (2), two main approaches for plants:

1. Agrobacterium - mediated gene transfer
2. Direct gene transfer

For (3), use promoter that will direct expression when

and where wanted may also require other
modifications such as removing or replacing introns.
*Both methods (Agrobacterium &
direct gene transfer) require
tissue culture system to
generate transgenic plants
 Most common method of engineering dicots, but also
used for monocots
Pioneered by J. Schell (Max-Planck Inst., Cologne)

Agrobacteria
soil bacteria, gram-negative, related to Rhizobia
species:
tumefaciens - causes crown galls on many dicots
rubi - causes small galls on a few dicots
rhizogenes - hairy root disease
radiobacter - avirulent
 Crown galls caused by
A. tumefaciens on
nightshade.

More about Galls:

http://waynesword.palomar.edu/pljuly99.htm
http://kaweahoaks.com/html/galls_ofthe_voaks.
html
In many ways, Agrobacterium, has been the most
successful method of delivering DNA into plants.
It is a naturally occurring plant pathogen. Complex
bacterium genome has been sequenced; 4
chromosomes; ~ 5500 genes
The species of choice for engineering dicot plants;
monocots are generally resistant
Some dicots more resistant than others (a genetic
basis for this)
It inserts DNA into the nucleus of a plant cell. The
inserted DNA contain genes that encode hormones
and food products for the bacteria to use to
support its own growth.
Here you can see the gall growth on the plant
tissue. This is the natural result of the infection.

Gall on
stem

Gall on
leaf
 Agrobacterium tumefaciens

Pili used for attachment to plant cells, and conjugation to

other bacteria.
Why use Agrobacterium?
Contains big plasmids :
Ti plasmid (A. tumefaciens)
Ri plasmid (A. rhizogenes)
Integration part of plasmid, T-DNA
encourage growth of tumour and roots
independently
T-DNA carries genes that encodes
hormones for hormone biosynthesis and
plant metabolites (opines & agropines) -
molecule containing N & C for energy
Plant host pathogen: 331 genera, 643
species
Chromosomal DNA
which is involved in the
inheritance functions
A circular DNA called
plasmid DNA.
The functions of these
DNA are separated
Plasmid DNA carries
the genes which is
involved in the tumour
or hairy root formation
in the plant cell.
Infection occurs at wound sites
Involves recognition and chemotaxis of the
bacterium toward wounded cells
Galls are real tumors, can be removed and
will grow indefinitely without hormones
Genetic information must be transferred to
plant cells
1. Synthesize a unique amino acid, called opine
Opine: octopine and nopaline (AA derivative)-
octopine derived from arginine and pyruvate,
nopaline derived from arginine and -
ketoglutarate.
agropine (sugar derivative)- derived from
glutamate
2. Opine depends on the strain of A. tumefaciens
3. Opines are catabolized by the bacteria, which can
use only the specific opine that it causes the plant
to produce.
4. Has obvious advantages for the bacteria, what
about the plant?
It was recognized early that virulent strains
could be cured of virulence, and that cured
strains could regain virulence when
exposed to virulent strains; suggested an
extra-chromosomal element.
Large plasmids were found in A.
tumefaciens and their presence correlated
with virulence: called tumor-inducing or Ti
plasmids.
1. Large (~200-kb)
2. Contain a vir region
3. Contain one or more T-DNA regions
4. Contain origin of replication
5. Contain a region eanabling conjugative transfer
6. ~10% of plasmid transferred to plant cell after
infection
7. Transferred DNA (called T-DNA) integrates semi-
randomly into nuclear DNA
8. Ti plasmid also encodes:
enzymes involved in opine metabolism
proteins involved in mobilizing T-DNA (Vir genes)
T-DNA is a segment of the plasmid which is
transferred into the plant cells and is thus
involved in the pathogenic effects on the
plants.
T-DNA is flanked by sequences which is
repeated 24 times on the right and left side.
This border will mark the position of the T-
DNA (LB & RB)
Ti plasmid of Nopaline strains: 1 T-DNA (20kb)
Octopine strains: 2 T-DNA regions TL (14kb) & TR
(7kb)
Only TL is oncogenic (contains aux & cyt genes)
TR contain genes for opine biosynthesis
T-DNA of nopaline Ti plasmids contains 13 ORFs,
TL DNA of octopine T-DNA contains 8 ORFs.
These ORFs have features of the eukaryotic, rather
than prokaryotic genes.
Both show extensive similarity in the core region
region that contains genes that code for proteins
involved in hormone biosynthesis (the oncogenes),
opine synthesis and for determining tumour size.
 Nopaline T-DNA / Octopine TL region

LB auxA auxB cyt tm1 ocs RB

LB, RB left and right borders (direct repeat)

auxA + auxB encodes enzymes in auxin biosynthesis
cyt encode isopentyl transferase (enzyme involved in
cytokinin production)
tm1 regulates tumor size
ocs (octopine synthase) - encodes opine synthesis

These genes have typical eukaryotic expression signals!

The oncogenes on T-DNA:

Genes that encode proteins involved in production

of auxin IAA (indole acetic acid):
auxA: tms 1 or iaaM encodes for triptophan
monooxigenase
auxB: tms 2 or iaaH encodes for indole 3-acetamide
hidrolyase;

Cyt: tmr gene or ipt - encodes isopentyl transferase

(catalyze the most important step in cytokinin
production).
 auxA auxB
Tryptophan indoleacetamide indoleacetic acid
(auxin)

cyt
AMP + isopentenylpyrophosphate isopentyl-AMP
(a cytokinin)

Increased levels of these hormones stimulate cell

division.

Explains uncontrolled growth of tumor.

1. On the Ti plasmid
2. Transfer the T-DNA to plant cell
3. Acetosyringone (AS) (a flavonoid) released by
wounded plant cells activates vir genes.
4. virA,B,C,D,E,F,G (7 complementation
groups, but some have multiple ORFs),
span about 30 kb of Ti plasmid.
virA - transports AS into bacterium, activates
virG post-translationally (by phosphoryl.)
virG - promotes transcription of other vir genes
virD2 - endonuclease/integrase that cuts T-
DNA at the borders but only on one strand;
attaches to the 5' end of the SS
virE2 - binds SS of T-DNA & can form channels
in artificial membranes
virE1 - chaperone for virE2
virD2 & virE2 also have NLSs (nuclear targeting
sequence), gets T-DNA to the nucleus of plant cell
virB - operon of 11 proteins, gets T-DNA through
bacterial membranes
(1) Signal recognition by Agrobacterium
(2) Attachment to plant cells
(3) Induction of vir genes
(4) T-strand production
(5) Transfer of T-DNA out of bacterial cell
(6) Transfer of T-DNA and Vir proteins into
plant cell and nuclear localization
When plant is wounded, it released phenolics (eg.
acetosyringone) and sugars signals perceived
by Agrobacterium.
Plant cell produce signaling molecules-
acetosyringone and hydroxyacetosyringone.
The wound signal indicates presence of
wounded cells, competent for transformation.
Controlled by 2 virulent loci in the Agrobacterium
chromosome, chvA & chvB
Intermediary binding when Agrobacterium is at
the wounded site
Involves 2 steps:
(1) Initial attachment via polysaccharide (product of
attR locus). a mesh of cellulose fibres is
produced by the bacterium.
(2) Action of chromosomal virulence genes (chvA and
chvB)
- chvA and chvB genes are involved in production and
secretion of cyclic -1,2-glycans and the pscA genes (psc:
polysaccharide) which are involved in secreting
succinoglycan.
- Agrobe strains carrying mutated chvA and chvB genes are
avirulent or extremely attenuated.
Operon vir is controlled by the constitutive
expression of VirA and G.
Vir A (a membrane-linked sensor kinase)
senses phenolics (eg. AS)
VirA autophosphorylates subsequently
phosphorylate & activate VirG.
VirG induces expression of all vir genes (eg.
virB,C,D,E)
Genes vir B,C,D,E needed for the excision of
T-DNA
vir D which encodes endonuclease.
Operon vir D has 4 reading frames:
VirD1 modulates VirD2 activity
VirD2 nicks T-DNA and directs T-DNA through
VirB/VirD4 transfer apparatus.
LB and RB recognized by VirD1-VirD2
complex VirD2 nicks the DNA at RB
(single-stranded nick) and attached to 5 end
of the displaced SS T-DNA strand.
Repair synthesis replaces the displaced strand
helped by VirC1
T-DNA-VirD2 complex is exported from the
Agrob cell by T-pilus (a membrane-channel
secretory system).
T-pilus is composed of proteins encoded by
virB operon and VirD4.
VirE2 and VirF are also exported from the
Agrob.
T-DNA-VirD2 complex and other Vir proteins
cross plants plasma membrane through
channels formed from VirE2.
Inside the cytoplasm, T-DNA strand is
covered with VirE2 proteins called as
mature T-complex.
VirE2 protects T-DNA from nuclease and
allow the correct conformation of T-DNA-
VirD2 complex to pass through nuclear pore
complex.
From Covey & Grierson
VirE2 may get DNA-protein complex across host plasma membrane

Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

 But Natures Agrobacterium
Has Problems
Infected tissues cannot be regenerated (via tissue culture)
into new plants
Why?
Phytohormone balance incorrect regeneration
Solution? Transferred DNA (T-DNA) modified by
Removing phytohormone genes
Retaining essential transfer sequences
Adding cloning site for gene of interest
 Monocots don't produce AS in response to
wounding.

Important: 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:

(1) Ti plasmids large, difficult to manipulate
(2) couldn't regenerate plants from tumors
Disarmed plasmid
Binary vector
Cointegrative vector
No oncogenic genes
Selectable marker genes
Reporter genes
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.
Binary vector system
Pbin19
Pgreen 0029
Homologous recombination between a co-integrative
vector and an intermediate cloning vector.
to introduce the DNA to be transferred to the plant cell, into a
modified T-DNA.
First, the DNA to be transferred is introduced into an
intermediate cloning vector based on an Escherichia coli
plasmid.

E. coli

(Gene of interest (DNA) is inserted here)

The cointegrative vector is a Ti plasmid from which the T-DNA
genes that encode oncogenic function have been removed and/or
replaced with a sequence that is also contained in the intermediate
vector.

Agrobacterium
The intermediate
vector that contains the Agrobacterium
foreign DNA is
introduced by
conjugation into
Agrobacterium that
contains the co-
integrative vector.

E. coli
The intermediate vector is not
stable in Agrobacterium.
Homologous recombination
between the intermediate vector HOMOLOGOUS
and the cointegrative vector RECOMBINATION
results in transferring the foreign
DNA to the cointegrative vector.
These vectors are designed so that once the foreign DNA, is
integrated into the cointegrative vector, it is located between its border
sequences.
 2 Common Transformation Protocols
1. Leaf-disc transformation - after selection and
regeneration with tissue culture, get plants with
the introduced gene in every cell

2. Floral Dip does not require tissue culture.

Reproductive tissue is transformed and the
resulting seeds are screened for drug-resistant
growth.
(Clough and Bent (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
Journal 16, 735743)
Making a transgenic
plant by leaf disc
transformation with
Agrobacterium.

S.J. Clough, A.F. Bent (1998) Floral dip: a simplified

method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant Journal 16, 735743.