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

Polarized Growth

 Axon and dendrite growth sites in neurons.

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

Vesicle docking and exocytosis

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

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

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.

Hypothesis Prediction

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.

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.

Produce GFP-AtSec8 Fusions

Plasmid sequencing

Stable transformation with Agrobacterium.

Agrobacterium transformation

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

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

Biolistics Particle Delivery

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

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?

Successful A. thaliana transformation

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

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