The Maize ropD Gene
Christine Neou
Dr. John Fowler
Botany 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
INACTIVE
Rop GDP
Rop GTP
ACTIVE

Rop GTPases in Signal Pathways
INACTIVE
Rop GDP
Rop GTP
ACTIVE
Binding of effector molecule

Rop GTPases in Signal Pathways
INACTIVE
Rop GDP
Rop GTP
ACTIVE
Binding of effector molecule
Signal for growth, differentiation or survival

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
Wild type Agarose gel of genotyping PCR
1 2 3 4
Lanes
1.
DNA ladder
2.
Wild type
3.
Homozygote
4.
Heterozygote
Homozygote
Heterozygote

Example: Genotyping of mc3 mutation
Wild type Agarose gel of genotyping PCR
1 2 3 4
Lanes
1.
DNA ladder
2.
Wild type
3.
Homozygote
4.
Heterozygote
Homozygote
Heterozygote

Example: Genotyping of mc3 mutation
Wild type Agarose gel of genotyping PCR
1 2 3 4
Lanes
1.
DNA ladder
2.
Wild type
3.
Homozygote
4.
Heterozygote
Homozygote
Heterozygote

Results of Genotyping
Mutation m1 m2 mc2 mc3 mc4
# genotyped # of homozygotes
52 0
15 1
10
37
9
1
8
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 magnification
Wild 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