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agrobacterium mediated gene transfer

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Title: 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.
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