AGROBACTERIUM TUMEFACIENS

MEDIATED GENE TRANSFER

IN PLANTS

SUSHANTA SARMA

M.Sc. BIOTECHNOLOGY

Agrobacterium - mediated Gene Transfer

Most common method of engineering dicots, but also used for monocots

Pioneered by J. Schell (Max-Planck Institute, Cologne)

Agrobacterium-

Soil borne, gram negative, rod shaped, motile found in rhizosphere

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 chemicals (sugar and

Phenolic molecules: acetosyringone) released from damaged plant cells

Contains Ti plasmid which can transfer its T-DNA region into genome of host plants

Agrobacterium tumefaciens

the species of choice for engineering dicot plants; monocots are generally

resistant.

some dicots more resistant than others (a genetic basis for this).

complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500

genes.

Infection and tumorigenesis

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.

Tumor characteristics

1. Synthesize a unique amino acid, called “opine”

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 features

Two strains of Ti-plasmid:

-Octopine strains- contains two T-DNA region: TL (14 kb) and TR ( 7 kb)

-Nopaline strains- contain one T-DNA region(20 kb)

Size is about 200 kb

Has a central role in Crown-gall formation

Contains one or more T-DNA region that is integrated into the genome of

host plants

Contain a vir region ~ 40 kb at least 8~11 vir genes

Has origin of replication

Contains a region enabling conjugative transfer

Has genes for the catabolism of opines

Ti Plasmid

(14 bp repeat)

(7 bp repeat)

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.

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.

Generation of the T-strand

overdrive

Right Border

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 border

Left border

DvirD/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 synthesis3. Integration into plant chromosome

Right border

Left border

D

T-strand coated with virE

T-DNA

virD nicks at Left Border sequence

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

Important points:

Binary vector system:

Strategy: Move T-DNA onto a separate, small plasmid.

Remove aux and cyt genes.

Insert selectable marker (kanamycin

resistance) gene in T-DNA.

Vir genes are retained on a separate plasmid. Put foreign gene between T-DNA borders. Co-transform Agrobacterium with both

plasmids. Infect plant with the transformed bacteria.

Binary vector system:

Practical application of

Agrobacterium-mediated plant

transformation:

Agrobacterium mediated transformation methods

are thought to induce less rearrangement of the

transgene.

Lower transgene copy number that direct DNA

delivery methods.

Successful production of transgenic plants

depends on the suitable transformation

protocols.

Recent research

Conclusion:

Agrobacteria are biological vector for introduction of

genes into plants. Agrobacterium-mediated

transformation is not restricted to eukaryotes as

Agrobacterium is also able to act on the gram positive

bacterium Streptomyces lividans. Agrobacterium can

transfer not only DNA but also proteins to the host

organisms through its type four secretion system.

References Bevan, M. (1984) “Binary Agrobacterium vectors for plant transformation”. Nucleic

Acids Res 12: 8711-8721.

Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell J., Van Montagu M. and

Leemans J. (1985) “Efficient octopine Ti plasmid-derived vectors for Agrobacterium-

mediated gene transfer to plants”. Nucleic Acid Research 13:4777-4788.

Chilton, M.D. (1983) “A vector for introducing new genes into plants”. Scientific

American, 248, 50-9.

Nadolska-Orczyk, A., Orczyk, W. and Przetakiewicz, A. (2000) “Agrobacterium-

mediated transformation of cereals – from technique development to its

application”. Acta Physiologiae Plantarum, 22, 77-8.

Kakkar, A. and Verma, V.K.,(2011) Agrobacterium mediated biotransformation.

Journal of Applied Pharmaceutical Science 01 (07); 2011: 29-35.