agrobacterium mediated gene transfer

1
AGROBACTERIUM TUMEFACIENS MEDIATED GENE
TRANSFERIN PLANTS

• SUSHANTA SARMA
• M.Sc. BIOTECHNOLOGY

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

• Synthesize a unique amino acid, called opine
• octopine and nopaline - derived from arginine
• agropine - derived from glutamate
• Opine depends on the strain of A. tumefaciens.
• 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 811 vir
  genes
• Has origin of replication
• Contains a region enabling conjugative transfer
• Has genes for the catabolism of opines

8
Ti Plasmid
(7 bp repeat)
(14 bp repeat)
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
Right Border
Left Border
T-DNA
overdrive
5
virD/virC
VirD nicks the lower strand (T-strand) at the
right border sequence and binds to the 5 end.
11
Generation of the T-strand
Left border
Right border
T-DNA
gap filled in
virE
T-strand
D
virD/virC
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
T-DNA
D
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
13
Overview of the Infection Process
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.

16
Binary vector system
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.
  

18
Recent research
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 134777-4788.
• 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.