The Maize ropD Gene Christine Neou Dr. John Fowler Botany and Plant Pathology

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The Maize ropD GeneChristine NeouDr. John
FowlerBotany and Plant Pathology
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Why use corn?

• Better understanding of how corn and other plants
  grow and develop

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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

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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

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G proteins - signaling molecules that bind GTP
Family
Ras Rho Rab Arf Ran
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G proteins - signaling molecules that bind GTP
Family
Ras Rho Rab Arf Ran
Subfamily
Rho Rac Cdc42 Rop
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G proteins - signaling molecules that bind GTP
Family
Ras Rho Rab Arf Ran
Subfamily
Rho Rac Cdc42 Rop
(Rho of Plants)
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Rop GTPases in Signaling Pathways
INACTIVE
Rop
GDP
Rop
GTP
ACTIVE
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Rop GTPases in Signal Pathways
INACTIVE
Rop
GDP
Binding of effector molecule
Rop
GTP
ACTIVE
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Rop GTPases in Signal Pathways
INACTIVE
Rop
GDP
Binding of effector molecule
Signal for growth, differentiation or survival
Rop
GTP
ACTIVE
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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

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The ropD genetic map
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Mutator Transposons
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Exons and Introns

• Exons - coding region
• Intron - sequences that are spliced out

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Goals

• Identify plants homozygous for the five alleles

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Goals

• Identify plants homozygous for the five alleles
• Characterize the five identified alleles by
  linking to a phenotype

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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.

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Genotyping by PCR

• DNA extraction
• Polymerase Chain Reaction (PCR)
• 3 primers used
• 2 gene specific primers (GSP)
• Mu primer

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Genotyping by PCR

• GSP
• DF3 located upstream of mutation

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Genotyping by PCR

• GSP
• DF3 located upstream of mutation
• DR5 located downstream of mutation

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Genotyping by PCR

• GSP
• DF3 located upstream of mutation
• DR5 located downstream of mutation
• Mu anneals to inverted repeats of transposon

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Example Genotyping of mc3 mutation
Agarose gel of genotyping PCR
Wild type
1 2 3 4

• Lanes
• DNA ladder
• Wild type
• Homozygote
• Heterozygote

Homozygote
Heterozygote
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Example Genotyping of mc3 mutation
Agarose gel of genotyping PCR
Wild type
1 2 3 4

• Lanes
• DNA ladder
• Wild type
• Homozygote
• Heterozygote

Homozygote
Heterozygote
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Example Genotyping of mc3 mutation
Agarose gel of genotyping PCR
Wild type
1 2 3 4

• Lanes
• DNA ladder
• Wild type
• Homozygote
• Heterozygote

Homozygote
Heterozygote
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Results of Genotyping
Mutation genotyped of homozygotes
m1 52 0
m2 15 1
mc2 10 1
mc3 37 8
mc4 9 1
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Example Phenotypes
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Epidermal cells of leaf tissue

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

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Epidermal cells of leaf tissue
Wild type - mostly straight rows, very few
areas of disorganization
Homozygote - larger areas of
disorganization
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Epidermal cells at high magnification
Wild type
Homozygote
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RNA

• Mature RNA contains only exons
• RNA cDNA
• Successful extraction of RNA from one sample

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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

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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

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Special Thanks to

• Howard Hughes Medical Institute
• National Science Foundation
• John Fowler and Lab