A unique bacterial species used for Plant-Fungal-Animal Transformation

1. What determines the choice vector?What determines the choice vector?
• Insertion size
• Vector size
• Restriction sites
• Copy number
• Cloning efficiency
• Ability to screen for inserts
• What downstream experiments do you want

2. Agrobacterium
A unique bacterial species used for
Plant-Fungal-Animal Transformation

3. Agrobacterium
• Gram -
• A natural genetic engineer
• 2 species
– A.tumefaciens (produces a gall)
– A. rhizogenes (produces roots)
• Dicots
• Worldwide

4. 1. Nopaline plasmids: carry gene for synthesizing
nopaline in the plant and for utilization (catabolism) in
the bacteria. Tumors can differentiate into shooty
masses (teratomas).
2. Octopine plasmids: carry genes(3 required) to
synthesize octopine in the plant and catabolism in the
bacteria. Tumors do not differentiate, but remain as
callus tissue.
3. Agropine plasmids: carry genes for agropine synthesis
and catabolism. Tumors do not differentiate and die
out.
Ti plasmids can be classified according toTi plasmids can be classified according to
the opines producedthe opines produced

5. Ti plasmid of A. tumefaciens

6. vir genes
opine catabolism
pTipTi
tra
for
transfer to
the plant
bacterial
conjugation
Agrobacterium chromosomal DNAAgrobacterium chromosomal DNA
chvA chvB
pscA
oriV
T-DNA-inserts into plant
genome

7. 1. 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.
2.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.
3.T-DNA encodes genes for opine synthesis and for tumor
production.
4. occ (opine catabolism) genes carried on the pTi and allows the
bacterium to utilize opines as nutrient.
Ti plasmids and the bacterial chromosomeTi plasmids and the bacterial chromosome
act in concert to transform the plantact in concert to transform the plant

8. Overview of the Infection Process

9. The infection process:
1. Wounded plant cell releases phenolics and nutrients.
2. Phenolics and nutrients cause chemotaxic response of A. tumefaciens
3. Attachment of the bacteria to the plant cell.
4. Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene
transcription and allow for T-DNA transfer and integration into plant chromosomal DNA.
5. Transcription and translation of the T-DNA in the plant cell to produce opines (food) and
tumors (housing) for the bacteria.
6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use
opines as a C, H, O, and N source.
Figure 18.2 and 18.3
Ti plasmid structure and
function. Note the wound-
induced plant phenolics
induce the vir genes on
the Ti plasmid.

10. TheThe virvir region is responsible for the transfer of T-region is responsible for the transfer of T-
DNA to the wounded plant cell.DNA to the wounded plant cell.
receptor
for acetyl-
syringone
positive
regulator
for other
vir genes
virA
constitutive
virG
virA is the sensor.
membrane
activated virG
Note: activated virG
causes its own promoter
to have a new start point
with increased activity.

11. virA is the sensor.
bacterial
membrane
Acetylsyringone is
produced by wounded
plant cells (phenolic
compound).
triggers auto-
phosphorylation
of virA
1 2
P
3
virG
virA
virG activates
transcription
from other vir
promoters.
VirA phosphorylates
virG which causes virG
to become activated.
virG is the effector.
Asg
Asg
P

12. Generation of the T-strandGeneration of the T-strand
overdrive
Right
Border
Left Border
T-DNA
virD/virC
VirD nicks the lower strand (T-strand) at the right
border sequence and binds to the 5’ end.
5’

13. Generation of the T-strandGeneration of the T-strand
Right borderLeft border
D
virD/virC
gap filled in
T-strand
T-DNA
virE
1. Helicases unwind the T-strand which is then coated by the virE
protein.
2. ~one T-strand produced per cell.

14. 1. Transfer to plant cell.
2. Second strand synthesis
3. Integration into plant chromosome
Right borderLeft border
D
T-strand coated with virE
T-DNA
virD nicks at Left Border sequence

16. VirE2 may get DNA-protein complex across host PM
Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

17. TheThe virvir region is responsible for the transfer of T-region is responsible for the transfer of T-
DNA to the wounded plant cell.DNA to the wounded plant cell.
ssDNA
binding
protein.
Binds T-
strand.
virA virGvirB
virC
virD virE
sensor effector
endo-
nucleas
e nicks
T-
DNA
Binds
overdrive
DNA.
membrane
protein; ATP-
binding
Note: The virA-virG system is related to the EnzZ-OmpR
system that responds to osmolarity in other bacteria.

19. (a) The pilus has not contacted the surface of the
recipient plant cell and the apparatus is unable to
transport T-complex.
(b) The pilus has contacted a receptor (?) on the surface
of the recipient plant cell. This induces the VirB
transporter, perhaps via a change in conformation, so
that it is now competent to transfer the T-complex to the
plant cell cytoplasm.
OM, outer membrane; IM, inner membrane; CW, plant
cell wall; PM, plasma membrane.
Model for contact-dependent activation of the
T-complex transport apparatus

20. Agrobacterium and genetic
engineering:
Engineering the Ti plasmid

21. Agrobacterium tumefaciens
• How is T-DNA modified to allow genes of
interest to be inserted?
– In vitro modification of Ti plasmid
• T-DNA tumor causing genes are deleted and replaced with
desirable genes (under proper regulatory control)
• Insertion genes are retained (vir genes)
• Selectable marker gene added to track plant cells
successfully rendered transgenic [antibiotic resistance
gene  geneticin (G418) or hygromycin]
• Ti plasmid is reintroduced into A. tumefaciens
• A. tumefaciens is co-cultured with plant leaf disks under
hormone conditions favoring callus development
(undifferentiated)
• Antibacterial agents (e.g. chloramphenicol) added to kill A.
tumefaciens
• G418 or hygromycin added to kill non-transgenic plant cells
• Surviving cells = transgenic plant cells

22. Co-integrative and binary vectors
Binary vector
LB RB
Co-integrative

25. Plant genetic engineering with
the binary Ti plasmid system
Clone YFG (your favorite gene) or
the target gene in the small T-DNA
plasmid in E. coli, isolate the plasmid
and use it to transform the disarmed
A. tumefaciens as shown.
Transgenic
plant
(disarmed)
Disarmed
Ti plasmid

26. MiniTi T-DNA based vector for plants
1.1. Binary vectorBinary vector: the: the virvir genesgenes
required for mobilization andrequired for mobilization and
transfer to the plant reside on atransfer to the plant reside on a
modified pTimodified pTi..
2. consists of the2. consists of the right and leftright and left
border sequencesborder sequences, a, a selectableselectable
markermarker (kanomycin resistance)(kanomycin resistance)
and aand a polylinkerpolylinker for insertion offor insertion of
a foreign gene.a foreign gene.
Disarmed vectors: do not produce tumors; can be
used to regenerate normal plants containing the
foreign gene.
miniTi

27. MiniTi T-DNA based vector for plants
modified Ti plasmid
a binary vector system
oriVoriV
virvir
T-DNA deleted
2
LB
RB
oriori
kanr
polylinker
miniTiminiTi
bombom1
bom = basis of mobilization

28. Transfer of miniTi from E. coli to
Agrobacterium tumefaciens
Triparental mating:Triparental mating:
bombom site forsite for
mobilizationmobilization
miniTi;miniTi;
kan resistancekan resistance
E. coli
Agrobacteriumstr resistant
pRK2013;pRK2013;
kan resistancekan resistance
contains tratra genes
modified pTimodified pTi
15A ori;15A ori;
E. coli or Agrobact.E. coli or Agrobact.
ColE1 oriColE1 ori
tra
bom
Ti oriVTi oriV

29. Steps in the mating 1-2:
Triparental mating:Triparental mating:
pRK2013;pRK2013;
kan resistancekan resistance
contains tratra genes
tra
ColE1 oriColE1 ori
bom
tra
1
2
E. coli
Helper plasmid (pRK2013) mobilizes itself
into 2nd
E. coli strain containing miniTi.
miniTi;miniTi;
kan resistancekan resistance

30. Steps in the mating 2-3:
E. coli
miniTi;miniTi;
kan resistancekan resistance
Agrobacterium
Helper plasmid mobilizes itself and the miniTi into Agrobacterium.
2 miniTi
3
pTi
pRK2013
miniTi
pRK2013 can
not replicate.
pRK2013

31. Selection of Agrobacterium containing the
miniTi on strep/kan plates
miniTi;miniTi;
kankan resistanceresistance
pRK2013;pRK2013;
kankan resistanceresistance modified pTimodified pTi
Agrobacterium
str resistant
Agrobacterium
str resistantplate on str and kan media
tra
str r
bom
can not replicate
pTi
miniTi
pRK2013
kanr
str r

33. 33
References:
1.Www. Wikipedia.org.in
2.Gustavo A. de la Riva1 , Joel González-Cabrera , Roberto Vázquez-Padrón ,
Camilo Ayra-Pardo (1998)Agrobacterium tumefaciens: a natural tool for plant
transformation. *. Journal of Bacteriology
3. Baron, C., Llosa, M., Zhou, S. and Zambryski, P.C.(1997). VirB1, a component
of the T-complex transfer machinery of Agrobacterium tumefaciens is processed to
a C-terminal secreted product VirB1*. Journal of Bacteriology 179:1203-1210.
4. Beaupre, C.E., Bohne, J., Dale, E.M. and Binns, A.N. (1997). Interactions
between VirB9 and VirB10 proteins involved in the movement of DNA from
Agrobacterium tumefaciens into plant cells. Journal of Bacteriology 179:78-89.
5. Binns, A.N. and Thomashow, M.F. (1988). Cell biology of Agrobacterium
infection and transformation of plants. Annual Review of Microbiology 42: 575-606.