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At the Plant/Agrobacterium Interface: Chemical Approaches to Signal Perception

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

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At the Plant/Agrobacterium Interface: Chemical Approaches to Signal Perception

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  1. At the Plant/Agrobacterium Interface: Chemical Approaches to Signal Perception Nora Goodman, Justin Maresh, Jin Zhang, David Lynn Emory University, Atlanta, GA

  2. 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

  3. Xenognosins • Agrobacterium relies on signals from the host for vir gene induction: 1) phenolics 2) monosaccharides 3) acidic pH

  4. 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

  5. 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

  6. Defining the Inhibition Model

  7. Inhibition Model This model will be tested, focusing on the reversibility and competitiveness of the inhibitors.

  8. β-galactosidase Assays Miller Units = C x Abs420 nm Abs600 nm x time

  9. HYDI Inhibition Curve

  10. Inhibition Model Test the reversibility of the inhibitor with washing assays. Data inconclusive.

  11. HYDI concentration = 0 uM HYDI concentration = 5 uM Tests for Inhibitor Reversibility Another test for reversibility: test the ability of AS to recover activity with concentration [HYDI] = 0 μM: Km = 2 μM Vmax = 2032 [HYDI] = 5 μM: Km = 6 μM Vmax = 930

  12. Inhibition Model Test the competitiveness by changing the K.

  13. Sugar Effect with AS Km = 4 μM Vmax = 4200 Km = 55 μM Vmax = 1100

  14. Effect of Sugar on HYDI inhibition IC50 = 24 μM Vmax = 1700 IC50 = 12 μM Vmax = 500

  15. Effect of Sugar on HF Inhibition IC50 = 33 μM Vmax = 2675 IC50 = 24 μM Vmax = 400

  16. Sugar Effect on ASBr IC50 = 21 μM Vmax = 3200 IC50 = 14 μM Vmax = 385

  17. 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

  18. Current model for ASBr binding The existing model of inhibition is flawed. Current model for HYDI and HF binding

  19. Test for Amine Binding • Ketone-containing compounds were synthesized No inhibition

  20. Inhibition Model The inhibitor must be binding to a site other than the phenolic binding site.

  21. Dr. David Lynn Dr. Vince Conticello Dr. Stefan Lutz The Lynn Lab Dr. Ken Walsh Dr. Lizhi Liang Justin Maresh Rong Gao Kun Lu Jijun Dong Peng Liu Fang Fang Andrew Palmer Hsiao-Pei Liu Yan Liang Brooke Rosenzweig Kaya Erbil Acknowledgements

  22. Latent Aldehyde MDIBOA contains a latent aldehyde: A series of analogs were tested; an aldehyde is required for inhibitory activity.

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