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 Cdc42Rop

G proteins - signaling molecules that bind GTP Family Ras Rho Rab Arf Ran Subfamily Rho Rac Cdc42Rop (Rho of Plants)

Rop GTPases in Signaling Pathways Rop GDP GTP INACTIVE ACTIVE

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

Rop GTPases in Signal Pathways Binding of effector molecule Signal for growth, differentiation or survival 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

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

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

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

Results of Genotyping Mutation# genotyped# of homozygotes m1 520 m2 151 mc2 101 mc3 378 mc4 91

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 typeHomozygote

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