Mutations in Arabidopsis Exocyst Gene AtSEC8

Jennie HinesMentor: 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• Thought to direct the way in which

cells grow

The exocyst in yeast

• Polarized cell growth in yeast• An 8 protein complex

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

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.

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.

The wild-type gene

T-DNA

T-DNA

Bam!

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

Part One:Initial Genotyping

DNA was extracted from the plants and PCR was done to find out which plants had the insert

Primers

From http://signal.salk.edu/tdnaprimers.html

LBb1

R F

T-DNA

Genome

~900 bp

~600 bp

900 bp

600 bp

Heterozygote Homozygous mutant

Homozygous wildtype

Results of Initial Genotyping

Homozygous mutants

Heterozygous mutants

Homozygous wild-type

F Plants 5 4 5

G Plants 1 6 10

Part Two:Crosses

The plants were crossed to see if the next generation had the mutation

Two Types of Crosses

• Self pollination• Backcrosses with

wildtypeTeeny tiny seeds that were planted one by one

Pretty Wild

From the AtSec8 “G” mutation self-cross

A Transmission Defect in the “G” Mutant

Homozygous mutants

Heterozygous mutants

Homozygous wildtype

F expected 5.5 11 5.5F Self-cross 3 11 8G expected 12 24 12G Self-cross 4 15 29

SIGNIFICANTLY DIFFERENT - p < 0.001

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.

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.

What next?

• Genotype the backcrosses• Study the pollen in media• Look for phenotypic differences

– Roots– Size of plants

Preliminary Results

Small homozygous mutantG mutant roots 0.3 cm longer

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.

Fin