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