1. Mutations in Arabidopsis Exocyst Gene AtSEC8 Jennie Hines Mentor: John Fowler

2. The Big Picture Plant morphology Cells have the ability to grow in specialized ways What the Fowler lab examines is the mechanism behind the way that cells grow Arabidopsis leaf cells

3. The Exocyst Thought to direct the way in which cells grow The exocyst in yeast Polarized cell growth in yeast An 8 protein complex

4. Pollen Tubes Pollen tubes exhibit polarized growth. One question is whether or not the exocyst is important for the pollen tube polarized growth. Picture thanks to Rex Cole pollen grain pollen tube

5. How does this apply to what I’m doing? My hypothesis is that the gene that codes for a part of the exocyst in Arabidopsis, AtSEC8, plays a role in the way pollen tubes grow. I have several different mutant plants, and the mutations may adversely affect AtSEC8. Prediction: The AtSEC8 mutant plants will have poorly functioning pollen.

6. Transmission Defects A mutation that causes pollen to function poorly also causes a transmission defect. Pollen grain Pollen tube Egg sac The mutation will be present in the next generation at a lower than expected frequency.

7. The wild-type gene T-DNA Bam!

8. F Mutations F and G G My mutations No defectTransmission defect 5’3’ AtSEC8 Other mutations previously studied Transmission defects F and G are in between

9. Part One: Initial Genotyping DNA was extracted from the plants and PCR was done to find out which plants had the insert

10. Primers From http://signal.salk.edu/tdnaprimers.html LBb1 RF T-DNA Genome ~900 bp ~600 bp

11. 900 bp 600 bp HeterozygoteHomozygous mutant Homozygous wildtype

12. Results of Initial Genotyping Homozygous mutants Heterozygous mutants Homozygous wild-type F Plants 545 G Plants 1610

13. Part Two: Crosses The plants were crossed to see if the next generation had the mutation

14. Two Types of Crosses Self pollination Backcrosses with wildtype Teeny tiny seeds that were planted one by one

15. Pretty Wild From the AtSec8 “G” mutation self-cross

16. A Transmission Defect in the “G” Mutant Homozygous mutants Heterozygous mutants Homozygous wildtype F expected 5.5115.5 F Self-cross 3118 G expected 122412 G Self-cross 41529 SIGNIFICANTLY DIFFERENT - p < 0.001

17. F Location Matters G No defectTransmission defect 5’3’ AtSEC8 5’ end have 0% transmission. 3’ end have 100% transmission. G is located between with 33% transmission.

18. Conclusions Since there was a transmission defect, it can be concluded that my “G” mutation in AtSEC8 likely causes problems with pollen tube growth. A New Hypothesis: The 3’ end of the gene (encoding the protein C-terminal end) is not critical for exocyst function.

19. What next? Genotype the backcrosses Study the pollen in media Look for phenotypic differences –Roots –Size of plants

20. Preliminary Results Small homozygous mutant G mutant roots 0.3 cm longer

21. Special Thanks to… John Fowler, Rex Cole, Zuzana Vejlupkova and Kirstin Arthur. And to the HHMI program for giving me the opportunity to be a part of this.

22. Fin