Transcript Agrobacterium tumefaciens: biology and biotechnology

Agrobacterium tumefaciens:
biology and biotechnology
• general introduction
• host recognition: the VirA/VirG two
component system
• macromolecular transport: the Type
IV secretion system (VirB complex)
• Agrobacterium and the creation of
transgenic plants
Strain T37
induced teratoma
on Kalenchoe
A. tumefaciens attached to plant
cells
EM of A. tumefaciens attached to
plant cell (courtesy L. Tilney)
Host recognition by
Agrobacterium tumefaciens
pTi
Nicotiana
tabacum
VirA and VirG are central to
the pathogenic process.
Pvir
VirA detects plant wound
signals and activates VirG
VirG binds vir operon
promoters at AT rich Vir boxes
and activates transcription of vir
gene operons.
virB
virC
virD
virE
virG
VirA-VirG,
a typical two-component
regulatory system
Inactive VirA Dimer
Activated VirA Dimer
Sugar-ChvE
H
ATP
AS and/or
phenol -binding
protein
Activated VirG
Low pH?
H-PO4
D
PO4
G
VirG-PO4 can activate
transcription of vir operons
VirA Domains/Functions
periplasmic
39-259
kinase
419-691
linker
279-418
receiver
712-829
E255L
TM1
TM2
H474
ChvE
phenolic
inducer
sugar
pH?
G1 G2
ATP
PO4
The E255L mutation reduces sugar-enhanced
perception of low concentrations of AS.
4500
4000
B-Galactosidase
3500
Wild type arabinose
Wild type glycerol
E255L arabinose
E255L glycerol
3000
2500
2000
1500
1000
500
0
0
1
10
uM AS
100
VirA Heterodimer
cell exterior
periplasm
E255L
X
cytoplasm
H
Q
Can co-expression of an inactive H474Q mutant and
the E255L mutant restore sugar-enhanced response to AS?
Initial Experiments expressed virA
alleles from multi-copy plasmids
1800
1600
1400
1200
1000
Wild type, glycerol
Wild type, arabinose
E255L, glycerol
E255L, arabinose
E255L/H474Q, glycerol
E255L/H474Q, arabinose
H474Q, arabinose
800
600
400
200
0
0
10
uM AS
100
Placement of a VirA mutant alleles
on a single copy vector
BssHII
PvirA
KpnI
BssHII
virA H474Q
(4.7 kb)
XbaI
virA E255L
KpnI
PstI HindIII
KpnI
lacZ
repC
Bla
pAW50
(6.9 kb)
ColE1
repA
repB
PvirA
BssHII
Sugar-enhanced response to AS is restored in
VirAE255L/H474Q heterodimers
E255L
E255L/H474Q
45
Activity of a
virB-lacZ fusion
in A348-3 (DvirA)
40
35
1200
glycerol
1000
30
arabinose
800
25
600
20
15
400
10
5
200
0
0
0
15
uM AS
30
0
2.5
uM AS
5
Future directions
•
•
•
Signal perception and integration
– pH sensing - where is this happening and how does it influence VirA?
Phenol perception - have yet to demonstrate that phenols actually bind VirA
– Role of the linker in signal transmission and/or perception (ratchet model)
Protein interactions
– Role of the periplasmic region in dimerization
– Interaction of ChvE with VirA - not understood in physical terms
– What is responsible for ‘multicopy’ negative effect
Role of other proteins: Gauri Nair has recently shown that the presence of a
2nd plasmid in Agro affects vir gene expression: need to identify genes
responsible for this
Macromolecular transfer from
Agrobacterium tumefaciens
• VirB complex as model for Type IV
secretion systems
• VirB-mediated protein translocation
from Agrobacterium to plant cell
VirB Homologues
VirB homologues are responsible for
natural transformation competence in
Helicobacter pylori
Agrobacterium tumefaciens
(from Smeets and Kusters 2002 Trends in Micriobiol. 10:159-162)
Helicobacter pylori
Expression of virE2 in planta
complements a virE2 mutant strain of
A. tumefaciens
Identification of a region of
VirE2 necessary for activity in
the bacterium but not the plant:
putative export signal?
Virulence of virE2 mutant strains
Strain
VirE2
A348
wild type
A348::virE2
none
A348::virE2 (pLS23)
N flag
A348::virE2 (pLS21)
C flag
A348::virE2 (pLS32)
D5
A348::virE2 (pMS13)
D13
A348::virE2 (pLS31)
D18
A348::virE2 (pMS14)
D23
A348::virE2 (pMS15)
D28
A348::virE2 (pMS16)
D33
A348::virE2 (pMS17)
D38
Virulence on
tobacco
+
+
+
-
Question: Do C-terminal
mutations block VirE2 transport
into plants?
Strategy: Create transgenic plants
producing various VirE2 mutant
proteins.
In planta complementation by
VirE2 C-terminal mutant forms
Functional domains of VirE2
Agrobacterium tumefaciens as a
tool for plant genetic engineering
• ‘disarming’ the T-DNA
• plant selectable markers
• generation of transgenic plants
• examples
Conserved (oncogenic) regions of
the T-DNA
Regeneration of transgenic
plantlets transformed by
“Disarmed” cointegrate vector
Regeneration of transgenic
tobacco plants transformed by
“Disarmed” cointegrate vector
Binary vectors for use in ‘disarmed’ A.
tumefaciens; this allows much easier cloning
and these vectors can replicate in both E. coli
and A. tumefaciens
Enhancer trap: take advantage of random TDNA insertion to identify organ/tissue/cell
specific regulatory elements
Left border of T-DNA
“minimal promoter”
Plant DNA
T-DNA
Plant enhancer drives Gal4 expression (from T-DNA) which
activates expression of GFP via the UAS regulatory element
Results: Identification of organ specific
regulatory sequences in Arabidopsis
A problem: lepidopteran larvae
eat crops!
Tomato stem pieces transformed
using Agro plus binary vector
carrying 35S-Bt toxin:
kanamycin selection
Regeneration of transgenic
tomato shoots
Flowering Bt-tomato plants
Leaf Bioassay: do tomato
hornworms eat Bt transgenic
tomato leaves
Vector plant
Bt plant
Whole plant cage bioassay set-up
Whole plant cage bioassay!!
Vector plant
Bt plant
Novel strategies in plant biotechnolgoy:
biodegradable plastics
Rhodobacter sphaeroides makes plastic polyhydroxybutyrate (PHB)!
PHB biosynthesis is a 3-step
pathway from existing precursor
(Acetyl CoA)
PHB biosynthetic genes from
Rhodobacter cloned and sequenced
These have been expressed in plants via introduction by A.
Tumefaciens. Results------------------------------------------>