elucidating the role of the protein vire3 in agrobacterium rhizogenes ability toinfect host cells...

Elucidating the Role of the Protein VirE3 in Agrobacterium rhizogenes’ Ability to

Infect 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.

gonorrhoeae1

Frequently used as a vector for creating GMOs4

5.34 billion pounds of crop increase3

46.6 million pounds of pesticide reduction3

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

1.2. VirE3

VirE3

Suicide Plasmid

3.

Project Description

10,oo0 bp6,000

bp4,000 bp

3,000 bp

2,000 bp1,500 bp

1,000 bp

VirE3

pRi1724

1,350 bpEco

RISal

Galls

VirE3

Project Description

HindIII

VirE3

Klenow Fragment

dNTPs

VirE3 VirE3

Mutated version of virE3

Creating the Mutated Form of VirE3

Project DescriptionCreating the Mutated Form of

VirE3

Suicide plasmid containing wild-type VirE3

HindIII

AGCTTAGCTTCTCGATGACGTTCGA

AGCTACTGCAAGCT AGCTTAGCTTTCGATGACGTTCGA TCGAATCGAA

Klenow FragmentdNTPs

AGCTACTGCAAGCTAGCTTAGCTTTCGATGACGTTCGATCGAATCGAA

AGCTACTGCA ATCGAA

G

Blunt End Ligation

HindIII digestion

Project Description Continued

VirE3

KanamycinR

AmpicillinR

VirE3

KanamycinR

AmpicillinR

E. coli

Project Description

Transformed E.coli

Project Description

VirE3

E. coli

VirE3

A.Rhizogenes

Electroporation

SacB

GentamicinR

Project Description Electroporation

Source: pnas.org

Low voltage pulse induces membrane pore formation

Project Description

VirE3

A. rhizogenes

HomologousRecombination

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, p703-714.

2. The FDA list of Completed Consultations of Bioengineered Foods. http://www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt=bioListing. Updated on April 30, 2011.

3. Tonouchi, Naoto. Merits and the Global Status of Genetically Modified (GM) Crops. Foods Food Ingredients J. Jpn., Vol. 210, No.7, 2005

4. 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. 5733-8.