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GEN 314

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Auxin. Cytokinin. Opine (nos) Ti-plasmid. Opine catabolism. Origin of replication. Virulence region ... aux = genes involved in the production of the auxin IAA ... – PowerPoint PPT presentation

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Title: GEN 314


1
GEN 314
  • Gene Manipulation Lecture Eight
  • Gene transfer to plants

2
DNA transfer into plant cells
  • Not the entire Ti-plasmid is transferred into
    plant cells
  • only a small specific segment of about 23 kb
    in size (Chilton et al. 1977)
  • termed transfer DNA (T-DNA)
  • T-DNA structure and organization has been well
    studied. Conclusions made (Thomashow et al. 1980
    Zambryski et al. 1980) include
  • integration can occur at many different sites,
    randomly in the plant nuclear genome nopaline
    plasmid T-DNA is simpler, i.e. single integrated
    segment (23 kb)
  • octopine plasmid T-DNA usually exists as two
    segments, i.e. the left segment (TL 14 kb) and
    the right segment (TR 7 kb)
  • TL includes genes necessary for tumour
    formation and TR encodes enzymes for agropine
    biosynthesis
  • gt TL usually exist as a single copy/cell (but up
    to 10 copies may also be found)
  • gt TR usually has high copy number

3
Nopaline plasmid T-DNA
  • 13 ORFs (see AJ237588 _at_ NCBI plasmid pTiC58
    T-DNA 22 ORFs!)
  • Two junctions
  • right-hand junction DNA sequence very
    precise
  • left-hand junction can vary by up to 100
    nucleotides
  • T-DNA is flanked by two (almost perfect) 25 bp
    direct repeat sequences border sequences
  • repeats are not transferred intact to the
    plant genome, as the T-DNA end-points lie
    internally within them
  • repeats are conserved between nopaline and
    octopine Ti-plasmids gt repeats are important in
    the T-DNA integration mechanism
  • however, left border can be removed without
    affecting tumour formation gt right border is
    essential (including sequences further to the
    right and adjacent to the repeat sequence) and
    left border has little or no transfer activity
    (Shaw et al. 1984)

4
Nopaline Ti-plasmid
5
Octopine Ti-plasmid
www.umanitoba.ca
6
Octopine plasmid T-DNA
  • TL-DNA 8 ORFs (e.g. pTiACH5/pTi15955!)
  • Also include two junctions,
  • (left right border sequences) overdrive
    or enhancer sequence (24 bp, assoc. with the
    right border seq. required for optimal T-DNA
    transfer)

Right border
Left border
cyt
ocs
aux
tm1
Slater et al. 2003
6a,b
5
2
1
4
7
3
Overdrive sequence
aux genes involved in the production of the
auxin IAA cyt encodes isopentyl transferase
involved in cytokinin production tm1 regulates
tumour size ocs octopine synthase
7
T-DNA transfer
  • Completed in several steps
  • attachment to wounded plant cells (bacteria
    produces polysaccharides cellulose fibres that
    attach to plant cells)
  • signal recognition by Agrobacterium (bacteria
    perceiving signals, such as phenolic substances
    sugars, released from wounded plant cells)
  • vir gene induction (a variety of virulence
    genes are involved see Table 3.1, Slater et al.
    2003)
  • T-strand production (excision of the T-DNA by
    virulence gene products)
  • transfer of T-DNA out of the bacterial cell
    (vir gene products bind to T-DNA to aid its
    transportation out of the cell through
    membrane-channels)
  • transfer of the T-DNA and vir proteins into
    the plant cell and nuclear localization
    (T-DNA/Vir protein complex mobilization through
    the NPC and nuclear localization involving
    interactions with plant cell proteins)

8
Virulence proteins on Ti plasmids
Table 3.1 Slater et al. (2003)
9
VirA (autophosphorylates) perceives signals of
phenolic substances sugars phosphorylates VirG
VirG induces the expression of the rest of vir
genes on the Ti plasmid. Also includes chvE,
which encodes sugar transporters that interact
with VirA.
Bacteria attaches to plant cells at a wound site
VirD1/VirD2 border sequence recognition. VirD2
creates nicks attach to 5-end of T-strand.
VirC1 recognize overdrive sequence
VirD2 VirE2 interact with plant proteins for
the nuclear import of mature T-complex
Immature T-complex is coated with VirE2 for
protection from plant nucleases (mature T-complex)
Tzfira and Citovsky (2006) Agrobacterium-mediated
genetic transformation of plants biology and
biotechnology. Curr. Opin. Biotech. 17 147 154
10
T-DNA uncoating
Tzfira and Citovsky (2006) Agrobacterium-mediated
genetic transformation of plants biology and
biotechnology. Curr. Opin. Biotech. 17 147 154
11
T-DNA uncoating and integration
  • Uncoating the T-DNA relies on the ability of the
    VIP1 to form a tertiary complex with VirE2 VirF
  • the F-box protein (VirF) targets the VIP1 and
    VirE2 to degradation and promote destabilization
    of VIP1 (this may involve proteins ASK1 and
    CULLIN) (Tzfira and Citovsky 2006)
  • T-DNA integration is not yet clear, but it is
    thought to involve the hosts DNA repair and
    packaging system
  • for now, it is assumed that the hosts DNA
    repair system converts (ss)T-DNA into dsT-DNA,
    which will then be recognized as broken DNA
    fragments gt integrated back into the genome
  • integration is suspected to be in the form of
    non-homologus end-joining (NHEJ) and not HR
    (homologous recombination)
  • recently, KU80 has been identified as the
    plant factor involved in T-DNA integration using
    the NHEJ pathway (Li et al. 2005)

12
End Lecture Eight
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