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