The Maize ropD GeneChristine Neou

Dr. John FowlerBotany and Plant Pathology

Why use corn?

Better understanding of how corn and other plants grow and develop

Why use corn?

Better understanding of how corn and other plants grow and develop

Learn mechanisms by which plants signal a response to stress or respond to disease

Why use corn?

Better understanding of how corn and other plants grow and develop

Learn mechanisms by which plants signal a response to stress or respond to disease

Use what we learn to perhaps breed plants that are better equipped to respond against stressors

G proteins - signaling molecules that bind GTP

Family

Ras

Rho

Rab

Arf

Ran

G proteins - signaling molecules that bind GTP

Family

Ras

Rho

Rab

Arf

Ran

Subfamily

Rho Rac Cdc42 Rop

G proteins - signaling molecules that bind GTP

Family

Ras

Rho

Rab

Arf

Ran

Subfamily

Rho Rac Cdc42 Rop(Rho of Plants)

Rop GTPases in Signaling Pathways

Rop

Rop

GDP

GTP

INACTIVE

ACTIVE

Rop GTPases in Signal Pathways

Binding of effector molecule

Rop

Rop

GDP

GTP

INACTIVE

ACTIVE

Rop GTPases in Signal Pathways

Binding of effector molecule

Signal for growth, differentiation or survival

Rop

Rop

GDP

GTP

INACTIVE

ACTIVE

The Role of Rops in Corn

???Function not known

Question: What is the role of Rops in plant growth and development?

At least 9 rops in corn

The ropD genetic map

Mutator Transposons

IR IR

Exons and Introns

Exons - coding region

Intron - sequences that are spliced out

Goals

Identify plants homozygous for the five alleles

Goals

Identify plants homozygous for the five alleles

Characterize the five identified alleles by linking to a phenotype

Goals

Identify plants homozygous for the five alleles

Characterize the five identified alleles by linking to a phenotype

Why homozygous plants?

They are the only plants that will exhibit a mutant phenotype.

Genotyping by PCR

DNA extraction

Polymerase Chain Reaction (PCR)

3 primers used: 2 gene specific primers

(GSP) Mu primer

Genotyping by PCR

GSP DF3 located upstream

of mutation

Genotyping by PCR

GSP DF3 located upstream

of mutation DR5 located

downstream of mutation

Genotyping by PCR

GSP DF3 located upstream

of mutation DR5 located

downstream of mutation

Mu anneals to inverted repeats of transposon

Example: Genotyping of mc3 mutation

1 2 3 4Lanes1. DNA ladder2. Wild type3. Homozygote4. Heterozygote

Agarose gel of genotyping PCR Wild type

Homozygote

Heterozygote

Example: Genotyping of mc3 mutation

1 2 3 4Lanes1. DNA ladder2. Wild type3. Homozygote4. Heterozygote

Agarose gel of genotyping PCR Wild type

Homozygote

Heterozygote

Example: Genotyping of mc3 mutation

1 2 3 4Lanes1. DNA ladder2. Wild type3. Homozygote4. Heterozygote

Agarose gel of genotyping PCR Wild type

Homozygote

Heterozygote

Results of Genotyping

Mutation # genotyped # of homozygotes

m1 52 0

m2 15 1

mc2 10 1

mc3 37 8

mc4 9 1

Example Phenotypes

Epidermal cells of leaf tissue

Wild type cells - mostly straight rows of cells with stomata spread evenly

Epidermal cells of leaf tissue

Wild type - mostly straight rows, very few areas of disorganization

Homozygote - larger areas of disorganization

Epidermal cells at high magnificationWild type Homozygote

RNA

Mature RNA contains only exons

RNA cDNA

Successful extraction of RNA from one sample

Conclusions

Observations have yielded no obvious mutant organismal phenotype

Epidermal cell experiments suggest a cell phenotype for homozygous plants

Preliminary data from RNA experiments are promising, experiments are still ongoing

The future…

Continue the experiments through the rest of the program and through the fall Continue looking for mutant phenotypes for

homozygous plants Use a computer program to analyze

epidermal cells from more plants Get more data from RNA

experiments

Special Thanks to

Howard Hughes Medical Institute

National Science Foundation

John Fowler and Lab