at the plant/agrobacterium interface: chemical approaches to signal perception
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At the Plant/Agrobacterium Interface: Chemical Approaches to Signal Perception. Nora Goodman, Justin Maresh, Jin Zhang, David Lynn Emory University, Atlanta, GA. Agrobacterium tumefaciens. Soil-borne bacterium responsible for Crown Gall Tumors - PowerPoint PPT PresentationTRANSCRIPT
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At the Plant/Agrobacterium Interface: Chemical Approaches
to Signal Perception
Nora Goodman, Justin Maresh, Jin Zhang, David Lynn
Emory University, Atlanta, GA
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Agrobacterium tumefaciens
• Soil-borne bacterium responsible for Crown Gall Tumors
• Transfers a piece of T-DNA from the Ti plasmid to the host plant causing production of tumors
• Currently the only known organism to routinely perform inter-kingdom gene transfer
• Used in transgenic plants
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Xenognosins
• Agrobacterium relies on signals from the host for vir gene induction:
1) phenolics
2) monosaccharides
3) acidic pH
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Acetosyringone (AS)
• Activation is thought to occur via the proton-transfer model with an amine as the base
• Induction is stronger with 2 methoxy groups, although it will take place with just 1 methoxy
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ASBr
• Design is based on the structure of AS• It was proposed that Br acts as a leaving
group, allowing nucleophilic attack on the α-carbon, which would make ASBr an irreversible inhibitor
NH2
A
H
O
OCH3
H3CO
O
H
A
H
A NH3
A
H
O
OCH3
H3CO
A
H
A
Br
O
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Defining the Inhibition Model
NH2
A
H
A
H
N
O
H3CO
A
H
A
H OCH3O
ReversibleInhibition
NO
H3CO
OCH3O
N
O
A
H
A
H
N
OMeO OH
OMe
O
MDIBOA
IC50 <1 M
OO OH
HF
IC50 = 25 M
O
MeO
OMe
O
OH
HYDI
IC50 = 5 M
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Inhibition Model
This model will be tested, focusing on the reversibility and competitiveness of the inhibitors.
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β-galactosidase Assays
Miller Units = C x Abs420 nm Abs600 nm x time
O
HO
HO
OH
HO
O
O2N
ONPG
-GalactosidaseO
HO
HO
OH
HO OH
O
O2N
Galactose Ortho nitrophenoxide
Yellow
ortho nitrophenyl--galatoside
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HYDI Inhibition Curve
0
100
200
300
400
500
600
0.1 1 10 100 1000
HYDI inhibition curve
Mill
er U
nits
Concentration (uM)
O
MeO
OMe
O
OH
HYDI
IC50 = 5 M
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Inhibition Model
Test the reversibility of the inhibitor with washing assays.Data inconclusive.
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Tests for Inhibitor ReversibilityAnother test for reversibility: test the ability of AS to recover
activity with concentration
0
500
1000
1500
2000
2500
3000
0 0.1 1 5 25
Varying AS with HYDI concentrations
Act
ivity
(M
ille
r U
nits
)
Concentration of HYDI (uM)
0
500
1000
1500
2000
2500
0.1 1 10 100 1000
HYDI inhibition of AS in Inducing Sugar
Act
ivity
(M
ille
r U
nits
)
AS Concentration (uM)
HYDI concentration = 0 uM
HYDI concentration = 5 uM
[HYDI] = 0 μM:
Km = 2 μM
Vmax = 2032
[HYDI] = 5 μM:
Km = 6 μM
Vmax = 930
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Inhibition Model
Test the competitiveness by changing the K.
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Sugar Effect with AS
0
1000
2000
3000
4000
5000
0.1 1 10 100 1000
Effect of Inducing Sugar with AS
AS concentration [uM]
-g
ala
ctos
ida
se a
ctiv
ity /
Mill
er
Un
it AS with inducing sugar
AS with non-inducing sugar
Km = 4 μMVmax = 4200
Km = 55 μMVmax = 1100
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Effect of Sugar on HYDI inhibition
0
500
1000
1500
2000
0.1 1 10 100
HYDI sugar comparison
Mill
er U
nits
concentratrion of inhibitor (uM)
HYDI with inducing sugar
HYDI with non-inducing sugar
IC50 = 24 μM
Vmax = 1700
IC50 = 12 μM
Vmax = 500
O
MeO
OMe
O
OH
HYDI
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Effect of Sugar on HF Inhibition
0
500
1000
1500
2000
2500
3000
0.1 1 10 100
HF sugar comparison
Act
ivity
(M
ille
r U
nits
)
Concentration of inhibitor (uM)
HF in inducing sugar
HF in non-inducing sugar
IC50 = 33 μM
Vmax = 2675
IC50 = 24 μM
Vmax = 400
OO OH
HF
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Sugar Effect on ASBr
0
500
1000
1500
2000
2500
3000
3500
0.1 1 10 100
ASBr Sugar Comparison
Act
ivity
(M
ille
r U
nits
)
Concentration of ASBr (uM)
ASBr in inducing sugar
ASBr in non-inducing sugar
50
100
150
200
250
300
350
400
0.1 1 10 100
ASBr in non-inducing sugar
Activ
ity (M
iller U
nits
)
Concentration of ASBr (uM)
IC50 = 21 μM
Vmax = 3200
IC50 = 14 μM
Vmax = 385
O
MeO
OMe
HOBr
ASBr
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Observations
• Increase in activity in inducing sugar
• Complete inhibition in both inducing and non-inducing sugar
• Virtually no shift in IC50
• When a shift was seen, IC50 was higher in inducing sugar: exact opposite of expected result
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The existing model of inhibition is flawed.
NH2
A
H
O
OCH3
H3CO
O
H
A
H
A NH3
A
H
O
OCH3
H3CO
A
H
A
Br
O
Current model for ASBr binding
Current model for HYDI and HF binding
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Test for Amine Binding
• Ketone-containing compounds were synthesized
MeO
OMe OMe
MeOMeO
OMe
ADIMBIC acid PEDIMBIC acid ADPE
O
OH
OH
O
O
OH
O
O
OH
No inhibition
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Inhibition Model
The inhibitor must be binding to a site other than the phenolic binding site.
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Acknowledgements
Dr. David Lynn
Dr. Vince Conticello
Dr. Stefan Lutz
The Lynn LabDr. Ken WalshDr. Lizhi LiangJustin MareshRong GaoKun LuJijun DongPeng LiuFang FangAndrew PalmerHsiao-Pei LiuYan LiangBrooke RosenzweigKaya Erbil
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Latent Aldehyde
MDIBOA contains a latent aldehyde:
A series of analogs were tested; an aldehyde is required for inhibitory activity.
N
OMeO OH
OMe
O
MDIBOA
N
OHMeO O
OMe
O
MDIBOA
Latent aldehyde
IC50 <1 M