Elucidating the Role of the Protein VirE3 in Agrobacterium rhizogenes Ability toInfect Host Cells Sarah Layoun Dr. Walt Ream Laboratory Source: International Society for Microbial Ecology

Only known prokaryote which can integrate segments of its DNA into eukaryotic DNA Soil bacteria known to infect a variety of plants About Agrobacterium Source: ag.ndsu.edu Crown Galls Disease Source: forestryimages.org Hairy Root Disease

Type IV Secretion System H. pylori, L. pneumophila, B. pertussis, N. gonorrhoeae 1 Frequently used as a vector for creating GMOs billion pounds of crop increase million pounds of pesticide reduction 3 About Agrobacterium

Method of Tumor Induction

Understanding the necessity of virE3 in T-DNA insertion and integration into host nucleus Comparing wild-type A. rhizogenes to virE3 mutants in ability to induce tumors Project Overview

Project Description VirE3 Galls VirE3 Suicide Plasmid 3.

Project Description 10,oo0 bp 6,000 bp 4,000 bp 3,000 bp 2,000 bp 1,500 bp 1,000 bp VirE3 pRi1724 1,350 bp EcoRISal Galls VirE3

Project Description HindIII Klenow Fragment dNTPs VirE3 Mutated version of virE3 Creating the Mutated Form of VirE3

Project Description Creating the Mutated Form of VirE3 Suicide plasmid containing wild-type VirE3 HindIII AGCTTAGCTTC TCGATGACGTTCGA AGCTACTGCAAGCT AGCTTAGCTT TCGATGACGTTCGA TCGAATCGAA Klenow Fragment dNTPs AGCTACTGCAAGCTAGCTTAGCTT TCGATGACGTTCGATCGAATCGAA AGCTACTGCA ATCGAAG Blunt End Ligation HindIII digestion

Project Description Continued Kanamycin R Ampicillin R Kanamycin R Ampicillin R E. coli

Project Description Transformed E.coli

Project Description E. coli A.Rhizogenes Electroporation SacB Gentamicin R

Project Description Electroporation Source: pnas.org Low voltage pulse induces membrane pore formation

Project Description A. rhizogenes Homologous Recombination A. rhizogenes

Project Description Arabidopsis thaliana source: nature.com Infect several plant species, including potatoes, carrots, and A. thaliana

Means of preventing plant diseases Improvement of GMO synthesis Possible insight into Type IV Secretion Systems Significance of Findings

Ernest and Pauline Jaworski Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Walt Ream Larry Hodges Acknowledgements

References 1. Fronzes, Rémi; Christie, Peter J.; Waksman, Gabriel. The Structural Biology of type IV secretion systems. Nature Reviews Microbiology, Oct2009, Vol. 7 Issue 10, p The FDA list of Completed Consultations of Bioengineered Foods. Updated on April 30, Tonouchi, Naoto. Merits and the Global Status of Genetically Modified (GM) Crops. Foods Food Ingredients J. Jpn., Vol. 210, No.7, Vaidya, M., Li, J., Krichevsky, A.,Citovsky, V. Uncoupling of the Functions of the Arabidopsis VIP1 Protein in Transient and Stable Plant Genetic Transformation by Agrobacterium. Proc Natl Acad Sci USA. April 2005, 102 (16) pg