Download Christine Neou Botany and Plant Pathology

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
Rop
GDP
GTP
ACTIVE
Binding of
effector
molecule
Rop GTPases in Signal Pathways
INACTIVE
Rop
Rop
GDP
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
Agarose gel of genotyping PCR
1 2 3
Lanes
1. DNA ladder
2. Wild type
3. Homozygote
4. Heterozygote
Wild type
4
Homozygote
Heterozygote
Example: Genotyping of mc3 mutation
Agarose gel of genotyping PCR
1 2 3
Lanes
1. DNA ladder
2. Wild type
3. Homozygote
4. Heterozygote
Wild type
4
Homozygote
Heterozygote
Example: Genotyping of mc3 mutation
Agarose gel of genotyping PCR
1 2 3
Lanes
1. DNA ladder
2. Wild type
3. Homozygote
4. Heterozygote
Wild type
4
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
Homozygote - larger areas of
few areas of disorganization
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