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
About Agrobacterium
∗ Only known prokaryote which can integrate segments of its
DNA into eukaryotic DNA
∗ Soil bacteria known to infect a variety of plants
Source: ag.ndsu.edu
Crown Galls Disease
Source: forestryimages.org
Hairy Root Disease
About Agrobacterium
∗ 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
Method of Tumor Induction
Project Overview
∗ 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 Description
1.
2.
VirE3
3.
VirE3
Galls
VirE3
Suicide Plasmid
Project Description
10,oo0 bp
6,000 bp
pRi1724
4,000 bp
3,000 bp
2,000 bp
VirE3
1,500 bp
Galls
1,000 bp
VirE3
EcoRI
1,350 bp
Sal
Project Description
Creating the Mutated Form of VirE3
HindIII
VirE3
VirE3
VirE3
Klenow Fragment
dNTPs
Mutated version of virE3
Project Description
Creating the Mutated Form of VirE3
HindIII
AGCTACTGCA AGCTTAGCTTC
TCGATGACGTTCGA ATCGAAG
Suicide plasmid containing
wild-type VirE3
Klenow Fragment
dNTPs
AGCTACTGCAAGCT AGCTTAGCTT
TCGATGACGTTCGA TCGAATCGAA
Blunt End Ligation
AGCTACTGCAAGCTAGCTTAGCTT
TCGATGACGTTCGATCGAATCGAA
HindIII digestion
Project Description Continued
VirE
VirE
KanamycinR
KanamycinR
AmpicillinR
AmpicillinR
E. coli
Project Description
Transformed E.coli
Project Description
VirE
SacB
Electroporation
GentamicinR
E. coli
VirE
A.Rhizogenes
Project Description
Electroporation
Low voltage pulse induces membrane
pore formation
Source: pnas.org
Project Description
Homologous
Recombination
VirE
A. rhizogenes
A. rhizogenes
Project Description
• Infect several plant species, including
potatoes, carrots, and A. thaliana
Arabidopsis thaliana
source: nature.com
Significance of Findings
∗ Means of preventing plant diseases
∗ Improvement of GMO synthesis
∗ Possible insight into Type IV Secretion Systems
Acknowledgements
∗
∗
∗
∗
∗
Ernest and Pauline Jaworski
Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. Walt Ream
Larry Hodges
References
1.
2.
3.
4.
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.
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.
Tonouchi, Naoto. Merits and the Global Status of Genetically Modified (GM)
Crops. Foods Food Ingredients J. Jpn., Vol. 210, No.7, 2005
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.