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
• 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
Xenognosins
• Agrobacterium relies on signals from the host for vir gene induction:
1) phenolics
2) monosaccharides
3) acidic pH
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
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
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
Inhibition Model
This model will be tested, focusing on the reversibility and competitiveness of the inhibitors.
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
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
Inhibition Model
Test the reversibility of the inhibitor with washing assays.Data inconclusive.
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
Inhibition Model
Test the competitiveness by changing the K.
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
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
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
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
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
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
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
Inhibition Model
The inhibitor must be binding to a site other than the phenolic binding site.
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
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
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