3. The bacterium that causes
crown gall disease in plants
has a natural vector for
transformation of desirable
traits from one plant to
another.
Plant Gene Transfer
via Agrobacterium
T-DNA
There are Two Major Methods of Plant Gene
Transfer
10. 3. Infects at root crown or just below the soil
line.
4. Can survive independent of plant host in the
soil.
5. Infects plants through breaks or wounds.
6. Common disease of woody shrubs,
herbaceous plants, particularly problematic
with many members of the rose family.
7. Galls are spherical wart-like structures
similar to tumors.
11. Opine Biosynthesis
1. Within tumor tissues, the synthesis of
various unusual amino acid-like compounds are
directed by genes encoded on the integrated
plasmid.
2. The type of opine produced is specified by
the bacterial T-DNA
3. Opines are used by the bacteria as a carbon
(nutrient) source for growth.
4. Opine catabolism within bacteria is
mediated by genes encoded on the Ti plasmid.
12. Only known natural example of DNA
transport between Kingdoms
1. (Virulent) strains
of A. tumefaciens
contain a 200-kb
tumor inducing (Ti)
plasmid
2. Bacteria
transfer a portion
of the plasmid DNA
into the plant host
(T-DNA).
T-DNA
13.
14.
15.
16.
17.
18.
19.
20. The T-DNA is transferred from the
Bacteria into the Nucleus of the Plant
1. Stably integrates (randomly) into the plant
genome.
2. Expression of genes in wild-type T-DNA
results in dramatic physiological changes to
the plant cell.
3. Synthesis of plant growth hormones
(auxins and cytokinins) neoplastic growth
(tumor formation)
21. Agrobacterium tumefaciens
plasmid DNA
Plasmid DNA is
cut open with
an enzyme.
chromosomal
DNA
A specific gene is
“cut out” of the
donor DNA using
the same enzyme.
New gene is
inserted into
the plasmid.
Plasmid is transformed
into Agrobacterium.
When mixed with plant
cells, Agrobacterium
duplicates the plasmid.
The new gene is transferred
into the chromosomal DNA
of the plant cell.
When the plant cell
divides, each daughter
cell receives the new
gene, giving the whole
plant a new trait.
25. Genes required to breakdownGenes required to breakdown opinesopines for use as afor use as a
nutrient sourcenutrient source are harbored on theare harbored on the Ti plasmidTi plasmid
in addition toin addition to virvir genes essential for thegenes essential for the
excisionexcision andand transporttransport of theof the T-DNAT-DNA to theto the
wounded plant cellwounded plant cell..
T-DNA
vir genes
opine catabolism
pTipTi
~200 kb~200 kb
tra
for transfer
to the plant
bacterial
conjugation
23 kb23 kb
26. 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.
Ti plasmids can be classified accordingTi plasmids can be classified according
to the opines producedto the opines produced
27. 3. Agropine plasmids: carry genes for agropine
synthesis and catabolism. Tumors do not
differentiate and die out.
CNH(CH2)2CHCO2H
NH
HO2C(CH2)2CHCO2H
H2N
HN
(Nopaline)
28. 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.
Ti plasmids and the bacterialTi plasmids and the bacterial
chromosome act in concert to transformchromosome act in concert to transform
the plantthe plant
29. 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.
31. 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’
32. Generation of the T-strandGeneration of the T-strand
Right
border
Left
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.
33. 1. Transfer to plant cell.
2. Second strand synthesis
3. Integration into plant chromosome
Right
border
Left
border
D
T-strand coated with virE
T-DNA
virD nicks at Left Border sequence
34. TheThe virvir region is responsible for the transferregion is responsible for the transfer
of T-DNA to the wounded plant cell.of T-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.
35. 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
36. TheThe virvir region is responsible for the transferregion is responsible for the transfer
of T-DNA to the wounded plant cell.of T-DNA to the wounded plant cell.
ssDNA
binding
protein.
Binds T-
strand.
virA virGvirB
virC
virD virE
sensor effector
endo-
nuclease
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.
37. 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’
38. Generation of the T-strandGeneration of the T-strand
Right
border
Left
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.
39. 1. Transfer to plant cell.
2. Second strand synthesis
3. Integration into plant chromosome
Right
border
Left
border
D
T-strand coated with virE
T-DNA
virD nicks at Left Border sequence
40.
41. 1. VirB1 may have local lytic activity that allows
assembly of the transporter at specific sites in
the cell envelope.
2. The processed VirB1*
peptide is secreted through
the outer membrane by an unknown mechanism.
3. The structural components of the pilus are VirB2
and VirB5.
4. Complexes of VirB7/9, formed by disulfide
bridges, may initiate assembly of the VirB channel.
5. The exact role of VirB3, 4, 6, 8, 10 and 11, and
VirD4 in the transporter apparatus is unknown.
Assembly of the Agrobacterium T-Complex
Transport Apparatus
42. 6. VirD4, VirB4 and VirB11 have nucleotide-binding
motifs that are essential for their activity.
7. The T-complex, consisting of a ss copy of T-DNA
bound to VirD2 and coated with VirE2, is exported
through the transport apparatus.
SP, signal peptide; SPI, signal peptidase I.
43.
44. (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
47. pTi-based vectors for plantpTi-based vectors for plant
transformation:transformation:
2. Early shuttle vectors integrated into the T-
DNA; still produced tumors.
1. Shuttle vector is a small E. coli plasmid using
for cloning the foreign gene and transferring to
Agrobacterium.
E. coli Agrobacterium
pTiShuttle plasmid
conjugation
50. Transformation of Arabidopsis plants
Dip floral buds
in 1 ml of
Agrobacterium
culture for 5 to
15 min.
Detergent added to allow
bacteria to infiltrate the
floral meristem.
51. Transformation of Arabidopsis plants
700 to 900
seeds per plant.
Germinate on
kanamycin plates
to select
transformants.
10 to 20
transformed
plants per plant.
10 day old seedlings
52. 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 residetransfer to the plant reside
on aon a modified pTimodified pTi..
2. consists of the2. consists of the right and leftright and left
border sequencesborder sequences, a, a
selectable markerselectable marker (kanomycin(kanomycin
resistance) and aresistance) and a polylinkerpolylinker
for insertion of a foreignfor insertion of a foreign
gene.gene.
Disarmed vectors: do not produce tumors; can be
used to regenerate normal plants containing the
foreign gene.
miniTi
53. 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
54. 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
Agrobacterium
str 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
55. 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
56. 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
57. 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
59. One way of physically introducing DNA into
cells is with a particlegun.
•Very tiny DNA-coated metal particles are
suspended in a drop on a macroprojectile.
•A discharge (from a gunpowder explosion or
from breakage of a membrane enclosing a
pressurized chamber) impels the
macroprojectile.
•The macroprojectile is stopped by a
stopping plate, but the microprojectiles
continue into the tissue below.
•The DNA introduced with the particles is
expressed
60.
61. 1. DNA- or RNA-coated gold/tungsten particles are
loaded into the gun and you pull the trigger.
Particle Bombardment using the Gene Gun
62. 2. A low pressure helium pulse delivers the coated
gold/tungsten particles into virtually any target
cell or tissue.
3. The particles carry the DNA cells do not have
to be removed from tissue in order to transform
the cells
4. As the cells repair their injuries, they integrate
their DNA into their genome, thus allowing for
the host cell to transcribe and translate the
transgene.
63. Agrobacteria are biological vectors for
introduction of genes into plants.
•Agrobacteria attach to plant cell
surfaces at wound sites.
•The plant releases wound signal
compounds, such as acetosyringone.
•The signal binds to virA on the
Agrobacterium membrane.
•VirA with signal bound activates virG.
Summary
64. •Activated virG turns on other vir genes,
including vir D and E.
•vir D cuts at a specific site in the Ti plasmid
(tumor-inducing), the left border. The left
border and a similar sequence, the right
border, delineate the T-DNA, the DNA that
will be transferred from the bacterium to
the plant cell
•Single stranded T-DNA is bound by vir E
product as the DNA unwinds from the vir D
cut site. Binding and unwinding stop at the
right border.
65. •The T-DNA is transferred to the
plant cell, where it integrates in
nuclear DNA.
•T-DNA codes for proteins that
produce hormones and opines.
Hormones encourage growth of the
transformed plant tissue. Opines feed
bacteria a carbon and nitrogen source.
