1. Sub-cellular Localization of Arabidopsis thaliana SEC8 In Polarly-Growing Cells Kory S. Herrick John E. Fowler, Ph.D. John E. Fowler, Ph.D.

2. Polarized Growth

3.  Axon and dendrite growth sites in neurons.

4. What is the exocyst?  Complex of eight proteins:  SEC3, SEC5, SEC6, SEC8, SEC10, SEC 15, EXO70, & EXO84.

5. Vesicle docking and exocytosis

6. Question  Is the exocyst complex associated with polarized cellular growth in plants?

7. Hypothesis A. thaliana SEC8 localizes asymmetrically to regions of the plasma membrane where polarized growth occurs.

8. Strategy  Generate plasmid constructs encoding Green Fluorescent Protein (GFP) fused to the AtSec8 gene.  Transformation of GFP-AtSec8 fusions into polarly-growing cells.  Characterization of gene product localization using fluorescence microscopy.

9. Hypothesis Prediction

10. General Overview Two experiments: 1.Long-term stable expression of GFP- labeled SEC8 in A. thaliana. 2.Short-term transient expression of GFP- labeled SEC8 in maize and Arabidopsis thaliana.

11. Experiment 1 Overview 1. Produce GFP-AtSec8 constructs. 2. Clone GFP-AtSec8 and GFP only control into pCamLAT52 Arabidopsis pollen vector. 3. Stable transformation into A. thaliana. 4. Look for GFP under fluorescence microscope.

12. Produce GFP-AtSec8 Fusions

13. Plasmid sequencing

14. Stable transformation with Agrobacterium.

15. Agrobacterium transformation

16. Experiment 1 Progress  Seeds harvested  Next steps:  Plant on hygromycin media, and select for transformed plants.  Characterize GFP-AtSEC8 localization in pollen.

17. Experiment 2  Transient transformation of GFP- AtSEC8 and GFP-only control plasmids into maize and A. thaliana cells.  Biolistic bombardment.

18. Biolistics Particle Delivery

19. Transformed maize cell:  Transmitted light:  Fluorescent light:

20. Experiment 2 Conclusions 1. GFP-tagged SEC8 did not demonstrate asymmetrical localization in maize. 2. Distribution suggests random intracellular diffusion 3. Problem with A. thaliana SEC8 in maize?

21. Successful A. thaliana transformation

22. Future Experimentation  Transiently transform GFP-AtSec8 into A. thaliana cells.  Characterize GFP-AtSec8 localization in stably-transformed plant.

23. Acknowledgments  John E. Fowler, Ph.D.  Kevin Ahern, Ph.D.  National Science Foundation.  Howard Hughes Medical Institute.  Center for Gene Research and Biotechnology.