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Mutations in Arabidopsis Exocyst Gene AtSEC8

Mutations in Arabidopsis Exocyst Gene AtSEC8. Jennie Hines Mentor: John Fowler. 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. The exocyst in yeast.

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Mutations in Arabidopsis Exocyst Gene AtSEC8

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  1. Mutations in ArabidopsisExocyst 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 in yeast The Exocyst • Thought to direct the way in which cells grow • 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. pollen grain pollen tube Picture thanks to Rex Cole

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

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

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

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

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

  11. 900 bp 600 bp Heterozygote Homozygous mutant Homozygous wildtype

  12. Results of Initial Genotyping

  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 SIGNIFICANTLY DIFFERENT - p < 0.001

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

  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 G mutant roots 0.3 cm longer Small homozygous mutant

  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

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