Agrobacteria
 soil bacteria, gram-negative, related to Rhizobia
 Causative agents of “Crown gall” disease of dicoltyledones
 Have ability transfer bacterial genes to plant genome
 Attracted to wound site via chemotaxis in response to
sugars and Phenolic molecules: acetosyringone released
from damaged plant cells
 Contains Ti plasmid which can transfer its T-DNA region
into genome of host plants

Agrobacteria
• soil bacteria, gram-negative, related to Rhizobia
Classification of Agrobacterium.
Two basis of classification
1. Basis of pathogenicity.
Agrobacterium tumefaciens- crown gall.
Agrobacterium rhyzogen- hairy root
Agrobacterium radiobacter- Avirulent
2. On growth pattern
Biotype I
Biotype II
Biotype III

History of Agrobacterium
• Smith & Townsend (1907)- said bacteria caused crown gall
disease
• Brown & Stonier (1958)-proposed that not whole bacteria but
some part of it causes disease
• Zenen et.al (1974)- noted virulent strain-Agrobacterium
tumefaciens
• Chilton et.al (1977)-reported Ti & Ri plasmid transfer to plant
causing disease

History of Agrobacterium
• Smith & Townsend (1907)- said bacteria caused crown gall
disease
• Brown & Stonier (1958)-proposed that not whole bacteria but
some part of it causes disease
• Zenen et.al (1974)- noted virulent strain-Agrobacterium
tumefaciens
• Chilton et.al (1977)-reported Ti & Ri plasmid transfer to plant
causing disease

Tumor characteristics
1. Synthesize a unique amino acid, called “opines”
•octopine and nopaline - derived from arginine
•agropine - 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.

Elucidation of the TIP (tumor-inducing
principle)
• 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.

Ti-plasmid
1. Large : 200-kb
2. Conjugative
3. ~10% of plasmid transferred
to plant cell after infection
4. Transfer DNA (called T-
DNA) integrates semi-
randomly into nuclear DNA
5. Contain a vir region ~ 40 kb
at least 8~11 vir genes
involved in mobilizing T-DNA
6. Has origin of replication
7. Has genes for the catabolism
of opines

Ti Plasmid
(14 bp repeat)
(7 bp repeat)

auxA auxB cyt ocs
LB RB
LB, RB – left and right borders (direct repeat)
auxA + auxB – enzymes that produce auxin
cyt – enzyme that produces cytokinin
Ocs – octopine synthase, produces octopine
T-DNA

• Two strains of Ti-plasmid:
Nopaline strains- contain
one T-DNA region(23 kb)
Octopine strains- contains
two T-DNA region: TL
(14 kb) and TR ( 7 kb)

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.

Ti plasmids and the bacterial chromosome act in concert
to transform the plant
• Agrobacterium tumefaciens chromosomal genes: chvA, chvB,
pscA required for initial binding of the bacterium to the plant
cell and code for polysaccharide on bacterial cell surface.
Chv A& B major role in exopolysaccharide production
psc A major role in T-DNA transport
chv E glucose and galactose transport
chvD, ilv, miaA and att, have virulence property.
• Virulence region (vir) carried on pTi, but not in the transferred
region (T-DNA).Genes code for proteins that prepare the T-
DNA and the bacterium for transfer.
• T-DNA encodes genes for opine synthesis and for tumor
production.
• occ (opine catabolism) genes carried on the pTi allow the
bacterium to utilize opines as nutrient.

Virulence genes.
• Virulence region consists of 24 genes in total
• Virulence genes are located in 8 operons from vir A-vir H
• vir A,F and G are monocistronic operons , where as vir B,C,D,E,H are
polycistronic
• virA - transports AS into bacterium, activates virG post-translationally
• virG –transcriptional activator; 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, gets T-DNA to the nucleus of
plant cell
• virB - operon of 11 proteins, conjugational pores between plant cell and
bacteria; gets T-DNA through bacterial membranes

Agrobacterium tumefaciens T-DNA transfer process
Steps of gene transfer in plant:-
1. Bacterial colonization
2. Induction of virulence system
3. Generation of T-DNA transfer complex
4. T-DNA transfer
5. Integration of T-DNA into plant.

Generation of the T-strand
overdrive
Right
Borde
r
Left
Border
T-
DNA
virD/virC
VirD nicks the lower strand (T-strand) at the
right border sequence and binds to the 5’ end.
5’

Generation of the T-strand
Right
bord
er
Left
borde
r
D
virD/virC
gap filled in
T-strand
T-DNA
virE
1. Helicases unwind the T-strand which is
then coated by the virE protein.
2. ~one T-strand produced per cell.

1. Transfer to plant cell.
2. Second strand synthesis
3. Integration into plant chromosome
Right
bord
er
Left
borde
r
D
T-strand coated with virE
T-DNA
virD nicks at Left Border sequence

VirE2 may get DNA-protein complex across host PM
Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

Overview of the Infection Process

 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

Limitations of Agrobacterium as cloning vector
Ti plasmids are large (approximately 200 to 800kb).--------remove large
segments of DNA that are not essential for a cloning vector
•The production of phytohormones by transformed cells growing in culture prevents
them from being regenerated into mature plants---remove cytokinin and auxin
genes
•A gene encoding opine synthesis is not useful to a transgenic plant and may lower the
final plant yield by diverting plant resources into opine production---- remove opine
synthesis gene
•Ti plasmid does not replicate in E.coli, the convenience of perpetuating and
manipulating Ti plasmids carrying inserted DNA sequences in that bacterium is not
available.
•Transfer of the T-DNA,which begins from the rightborder, does not always end at the
left border. Rather, vector DNA sequences past the left border are often transferred,
although the transfer of these sequences is not often tested for.

To overcome these constraints, Ti plasmid-based vectors are engineered which are
similarly organized and contain the following components.
• A selectable marker gene, such as neomycin phosphotransferase(npt), that confers
kanamycin resistance on transformed plant cells. Because the npt gene, as well as many
of the other marker genes used in plant transformation, is prokaryotic in origin, it is
necessary to put it under the control of plant (eukaryotic) transcriptional regulation
signals, including both a promoter and a termination–polyadenylation sequence, to
ensure that it is efficiently expressed in transformed plant cells.
•An origin of DNA replication that allows the plasmid to replicate in E. coli. In some
vectors, an origin of replication that functions in A.tumefaciens has also been added.
•The right border sequence of the T-DNA region(although most cloning vectors
include both a right and a left border sequence)
•A polylinker (multiple cloning site) to facilitate insertion of the cloned gene into
the region between T-DNA border sequences.
•A “killer” gene encoding a toxin downstream from the left border to prevent unwanted
vector DNA past the left border from being incorporated into transgenic plants. If this
incorporation occurs, and the killer gene is present, the transformed cells will not
survive.

Cointegrate Vector
The Cloning or intermediate vector has a plant
selectable marker gene, the target gene, the
right border, an E. coli origin of DNA
replication, and a bacterial selectable marker
gene.
The intermediate vector recombines with a
disarmed Ti plasmid that lacks both the tumor-
producing genes and the right border of the T-
DNA within A. tumefaciens.
The intermediate vector and the disarmed
helper Ti plasmid both carry homologous DNA
sequences that provide a shared site for in vivo
homologous recombination; normally these
sequences lie inside the T-DNA region.
Following recombination, the cloning vector
becomes part of the disarmed Ti plasmid,
which provides the vir genes necessary for the
transfer of the T-DNA to the host plant cells.

2) COINTEGRATED VECTOR

Binary vector system
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:
• E. coli and A. tumefaciens origins of DNA replication, i.e., an E. coli–A. tumefaciens
shuttle vector, or a single broad-host-range origin of DNA replication(RK2).
•Selectable marker genes for plants and bacteria
•T DNA border sequences
•Multiple cloning site

•All the cloning steps are carried out in E. coli before the vector is introduced into A.
tumefaciens. The T-DNA border sequences are sub cloned on a small E. coli plasmid.This
plasmid, called mini-Ti or micro-Ti, can be introduced into an Agrobacterium strain
•The recipient A. tumefaciens strain carries a disarmed Ti plasmid which acts as a helper
plasmid as it contains a complete set of vir genes enabling the T-DNA from the binary
cloning vector to be inserted into the plant chromosomal DNA.

1) BINARY VECTOR

Plant transformation based on
direct DNA delivery
1.Polyethylene glycol (PEG)-mediated
protoplast transformation.
2.Particle bombardment
3.Electroporation
4.Microinjection
5.Ultrasound mediated
6.Pollen-mediated
7.Cell penetrating peptides

Gene gun or Particle Bombardment or
Biolistic or Balistic method
Used for most of Monocotyledons
Usefullto introduce DNA into mostly all tissues:
•Shoot apical meristems
•Monocotyledons
•Leaf blades
•Immature and mature embryos
•Root and shoot sections
•Plant cell suspensioni
•Callus culture
•Pollen
•Mitochondria and chloroplast

Particle bombardment
PDS-1000
First particle gun was developed by Sanford and colleagues in
1987. They introduced CAT (chloremphenicol acetyl
transferase) gene into onion epidermal cells and detected
transient gene activity.
First transgenic plants were produced in 1988 using soybean
and tobacco tissue culture. Christou (1988) and Klein (1988)
bombarded soybean shoot meristem and tobacco leaf,
respectively. Christou recovered chimeric transgenic soybean
plants that transmitted the gene into next generation.
Particle bombardment is the method of choice for chloroplast
transformation.

METHOD
1. Precipitate DNA onto 1- 2 um tungsten or gold
particles.
2. Accelerate particles to high to velocities that
allow their entry into plant cells/nuclei
3.Accelaration is achieved by devices which vary
in design and function
•Pressurized helium gas
•Electrostatic energy released by a droplet of
water exposed to high voltage

Gold or tungsten particles are used
gold
tungsten
•Tungsten particles are cheaper but are of
irregular shape and size
•Toxic to certain cell types and show surface
oxidation leading to precipitation of DNA
•Tend to form aggregates after addition of
DNA which reduces paticle dispersion
•Gold particles are
uniform in size and
shape , show low
toxicity but are costlier
and show variable
coating of DNA

• Coating of DNA to microprojectiles is by
precipitation
• One approach is to mix 1.25 – 1.8 mg of
microparticles with 0.5 – 70 ug DNA in CaCl2
and spermidine solution
• Vortexed to ensure uniform coating
• After coating microprojectiles are transferred
to macrocarrier membrane , allowed to dry
and immediately used

The Helium Gas Gun – Circa 2000

DNA is bound to the microprojectiles, which impact the
tissue or immobilized cells at high speeds.
J. Sanford & T. Klein, 1988
Original 22-caliber biolistic gun

Helium particle gun

Gene Gun