Transcript Methods of profucing transgenic plants

Lecture 1&2
Agrobacterium mediated plant
transformation
A.Tumefaciens gall is not a tiny thing
Biology of A. tumefaciens
Well known to induce crown gall tumor
A.tumefaciens lives around
root surfaces (in rhizosphere)
where it using nutrients
that leak from the root tissues
infects only through wound sites
and actively chemotactic to them
www-genvagar.slu.se/teknik/ djup/plasm.htm
Bacterial T-plasmid produces
receptors for acetosyringone
Plant wound produces
acetosyringone
The basis of Agrobacteriummediated genetic engineering
T-DNA of A. tumefaciens is excised and integrates into
the plant genome as part of the natural infection
process.
Any foreign DNA inserted into the T-DNA will also be
integrated.
Important genes encoded by
Ti plasmid
1. Cytokinins
(plant hormone for cell plant division and tumorous growth)
2. Enzymes for indoleacetic acid (auxin) synthesis
Another plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and
preventing aging)
3. Enzymes for synthesis and release of novel plant
metabolites:
the opines (uniques amino acid derivatives)
the agrocinopines (phosphorylated sugar derivatives) .
Nopaline
Opines and agrocinopines are NUTRIENTS for A.tumefacies.
They can not be used by other bacterial species
It provides unique niche for A.tumefaciens
Cytokinins are plant hormones that are
derivatives of the purine adenine.
Zeatin is one of cytokinines which synthesis
may be encoded by Ti plamid
isolated from corn (Zea mays).
Cell specific expression of cytokinin
in the A.tumefaciens infected cell
Opines are nutrients that are also
for quorum sensing
The plant cells start to secrete
the opines
from transferred bacterial T DNA
opine diffuses
into the surrounding cells
and serves as a signal molecules
for the conjugation
of the agrobacterium
(Quorum sensing)
Ti Plasmid
T-DNA
region
DNA between
L and R borders is
transferred to plant
as ssDNA;
Tumorproducing
genes
Opine catabolism
Virulence region
ORI
T-DNA encoded
genes can be
substituted by
target genes
Agrobacterium
A unique bacterial species
Plant-Fungal-Animal Transformation
Agrobacterium tumefaciens
1. Soil bacterium closely related to Rhizobium.
2. Causes crown gall disease in plants (dicots).
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 problamatic
with many members of the rose family.
7. Galls are spherical wart-like structures
similar to tumors.
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 
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)
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.
Overview of the Infection Process
How is the signal recognition
(acetosyringone and other plant
phenolics) converted to gene
activation and other cellular
responses?
Bacterial 2-Component Signal Transduction
Systems
1. Component 1 : Sensor kinase
i) Substrate receptor, signal recognition
domain, input domain (periplasmic)
ii) Signal transduction domain, membrane
spanning region
iii) Autokinase domain, phosphorylation
domain (cytoplasmic)
a) ATP binding (sub) domain
b) phosphorylation-phosphotransfer
(sub)-domain
2. Component 2 : Response regulator
i) Phosphorylation domain
ii) DNA binding domain
Simplest case: transcriptional activator when
phosphorylated
First component is typically (auto)-phosphorylated
on a His residue and transfers to a Asp group on
the response regulator (second component).
The EnvZ/OmpR System of E. coli
Senses changes in extracellular osmolarity
Agrobacterium tumafaciens senses
acetosyringone via a 2-component-like
system
3 components: ChvE, VirA, & VirG
1. ChvE
periplasmic protein binds to sugars, arabinose,
glucose
binds to VirA periplasmic domain
 amplifies the signal
2. VirA : Receptor kinase
1. Membrane protein five functional domains:
a) Periplasmic binds ChvE-sugar complex does
NOT bind acetosyringone
b) Transmembrane domain
c) Linker region BINDS acetosyringone NOTE
this is on the cytoplasmic side!
d) Transmitter domain (His) auto- phosphorylates
and then transfers to the response regulator
protein VirG
e) Inhibitory domain  in absence of analyte will
bleed off the phosphate from the His in the
transmitter domain (to an Asp)
3. VirG : Response Regulator
a) Receiver domain that is phosphorylated on an
Asp residue by the His on the transmitter domain
of VirA
b) Activates the DNA binding domain to promote
transcription from Vir-box continaing promoter
sequences (on the Ti plasmid)
sugars
VirA
Periplasmic domain
ChvE
receiver
acetosyringone
Transmitter
Inhibitory domain
VirG
DNAbinding
Crown gall
tumors
a natural
example of
genetic
engineering.
Agrobacterium/plant
interactions
Agrobacterium at
wound site
transfers T-DNA
to plant cell.
opines
Agrobacterium in soil
use opines as nutrients.
Genes required to breakdown opines for use as a
nutrient source are harbored on the Ti plasmid
in addition to vir genes essential for the
excision and transport of the T-DNA to the
wounded plant cell.
T-DNA 23 kb
tra
vir genes
for transfer
to the plant
pTi
~200 kb
bacterial
conjugation
opine catabolism
Ti plasmids can be classified according
to the opines produced
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.
H2N
(Nopaline)
CNH(CH2)2CHCO2H
HN
NH
HO2C(CH2)2CHCO2H
Ti plasmids and the bacterial
chromosome act in concert to transform
the plant
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.
Agrobacterium chromosomal DNA
pscA
chvA chvB
T-DNA-inserts into plant genome
for
transfer
to the
plant
vir genes
pTi
tra
bacterial
conjugation
opine catabolism
oriV
Generation of the T-strand
Left
Border
Right
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.
Generation of the T-strand
Left
border
T-DNA
Right
border
gap filled in
virE
virD/virC
T-strand
D
1. Helicases unwind the T-strand which
is then coated by the virE protein.
2. ~one T-strand produced per cell.
Left
border
T-DNA
Right
border
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
The vir region is responsible for the transfer
of T-DNA to the wounded plant cell.
virA is the sensor.
activated virG
membrane
constitutive
virA
receptor
for acetylsyringone
virG
positive
regulator
for other
vir genes
Note: activated virG
causes its own promoter
to have a new start point
with increased activity.
1
Asg
virA is the sensor.
P
virA
Asg
bacterial
membrane
Acetylsyringone is
produced by wounded
plant cells (phenolic
compound).
3
VirA phosphorylates
virG which causes
virG to become
activated.
P
virG
2
triggers autophosphorylation
of virA
virG activates
transcription
from other vir
promoters.
virG is the effector.
The vir region is responsible for the transfer
of T-DNA to the wounded plant cell.
sensor
virA
effector
virB virG
virC
virD
virE
ssDNA
binding
protein.
Binds Tstrand.
membrane
endoBinds
protein;
overdrive nucleas
ATP-binding DNA.
e nicks
TDNA
Note: The virA-virG system is related to the EnzZOmpR system that responds to osmolarity in other
bacteria.
Generation of the T-strand
Left
Border
Right
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.
Generation of the T-strand
Left
border
T-DNA
Right
border
gap filled in
virE
virD/virC
T-strand
D
1. Helicases unwind the T-strand which
is then coated by the virE protein.
2. ~one T-strand produced per cell.
Left
border
T-DNA
Right
border
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
Assembly of the Agrobacterium T-Complex
Transport Apparatus
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.
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.
Model for contact-dependent activation of the Tcomplex transport apparatus
(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.
Locations of the Vir Protein Components
of the T-DNA transfer system
1. The VirB and VirD4 proteins are grouped
according to probable functions:
a) exocellular proteins mediating attachment
(VirB1*, VirB2 pilin and VirB5)
b) channel proteins (VirB3, VirB6, VirB7,
VirB8, VirB9 and VirB10)
c) ATPases (VirB4, VirB11 and VirD4).
Agrobacterium
can be used to
transfer DNA
into plants
pTi-based vectors for plant
transformation:
1. Shuttle vector is a small E. coli plasmid using
for cloning the foreign gene and transferring to
Agrobacterium.
2. Early shuttle vectors integrated into the TDNA; still produced tumors.
Shuttle plasmid
E. coli
conjugation
pTi
Agrobacterium
Several hundred
tumors containing
foreign gene can be
grown for
experimental
purposes.
Transformed
sunflower seedlings
3 weeks after
inoculation
Harvest time!
Transformation of Arabidopsis
plants
Detergent added to allow
bacteria to infiltrate the
floral meristem.
Dip floral buds
in 1 ml of
Agrobacterium
culture for 5 to
15 min.
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
Summary
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.
•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.
•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.