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

1. The Maize ropD GeneChristine NeouDr. John FowlerBotany and Plant Pathology

2. Why use corn? • Better understanding of how corn and other plants grow and develop

3. 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

4. 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

5. G proteins - signaling molecules that bind GTP Family Ras Rho Rab Arf Ran

6. G proteins - signaling molecules that bind GTP Family Ras Rho Rab Arf Ran Subfamily Rho Rac Cdc42 Rop

7. G proteins - signaling molecules that bind GTP Family Ras Rho Rab Arf Ran Subfamily Rho Rac Cdc42 Rop (Rho of Plants)

8. Rop GTPases in Signaling Pathways INACTIVE Rop GDP Rop GTP ACTIVE

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

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

11. 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

12. The ropD genetic map

13. Mutator Transposons

14. Exons and Introns • Exons - coding region • Intron - sequences that are spliced out

15. Goals • Identify plants homozygous for the five alleles

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

17. 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.

18. Genotyping by PCR • DNA extraction • Polymerase Chain Reaction (PCR) • 3 primers used: • 2 gene specific primers (GSP) • Mu primer

19. Genotyping by PCR • GSP • DF3 located upstream of mutation

20. Genotyping by PCR • GSP • DF3 located upstream of mutation • DR5 located downstream of mutation

21. Genotyping by PCR • GSP • DF3 located upstream of mutation • DR5 located downstream of mutation • Mu anneals to inverted repeats of transposon

22. 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

23. 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

24. 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

25. Results of Genotyping

26. Example Phenotypes

27. Epidermal cells of leaf tissue • Wild type cells - mostly straight rows of cells with stomata spread evenly

28. Epidermal cells of leaf tissue Wild type - mostly straight rows, very few areas of disorganization Homozygote - larger areas of disorganization

29. Epidermal cells at high magnification Wild type Homozygote

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

31. 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

32. 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

33. Special Thanks to • Howard Hughes Medical Institute • National Science Foundation • John Fowler and Lab