2. 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
3. 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.
4. 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.
5. 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.
6. 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.
7. 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
9. 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.
10. Generation of the T-strand
overdrive
Right
Border
Left
Border
T-DNA
virD/virC
5’
VirD nicks the lower strand (T-strand) at the
right border sequence and binds to the 5’ end.
11. Generation of the T-strand
Right
border
Left
border
T-DNA
D
virD/virC
gap filled in
T-strand
virE
1. Helicases unwind the T-strand which
is then coated by the virE protein.
2. ~one T-strand produced per cell.
12. Right
border
Left
border
D
T-DNA
T-strand coated with virE
virD nicks at Left Border sequence
1. Transfer to plant cell.
2. Second strand synthesis
3. Integration into plant chromosome
14. Important points:
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
15. 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.
17. 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.
19. 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.
20. 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.
