1. Purification of a Secreted Agrobacterium rhizogenes Protein(GALLS) Required for Gene Transfer to Plants
Josh Cuperus, Larry Hodges,
Dr. Walt Ream
Department of Microbiology
Oregon State University

2. Agrobacterium Infects plants by introducing new DNA
Integrates DNA into plant genome
Several species that have this ability
Very useful for genetic modification of plants

3. Crown Gall Disease
Caused by Agrobacterium tumefaciens

4. Agrobacterium tumefaciens plant infection and transformation
Ti plasmid
T-DNA Region
Bacterial Cell
Plant Cell
T-DNA integrated into plant genome

5. Plant D2 E2
Nucleus
Agrobacterium E1

6. Agrobacterium rhizogenes
Causes hairy root disease instead of crown gall.
DNA transfer occurs without two essential proteins (VirE1 and VirE2) found in Agrobacterium tumefaciens.

7. Arrangement of Virulence Operons in Ti & Ri Plasmids

8. Ri & Ti Plasmid Maps
Ti Plasmid
Ri Plasmid

9. GALLS Replaces VirE2 by Mixed Infection
A. tumefaciens D2
plant
nucleus D2
VirB/D4
Agrobacterium rhizogenes D2
VirB/D4
GALLS
GALLS

10. Domains in the GALLS Protein
NTP-Binding TraA-Like T4SS

11. Similarities between GALLS and VirE2
Both have a type four secretion signal.
Both contain a nuclear localization signal, however they are not the same amino acid sequence.

12. Differences between GALLS and VirE2
Nucleotide sequences show no homology.
Only GALLS has a nucleoside triphosphate binding motif.
GALLS has a molecular weight more than three times that of VirE2.
There is no evidence of a chaperone for GALLS.

13. Purpose Protein purification
Use in creating antibodies that will work on normal GALLS protein
Antibodies will allow recognition of protein in other studies

14. Polyhistidine affinity tags
Series of 6 histidine amino acid residues allows for purification because of its affinity for a nickel column, allowing most other proteins to be removed.

16. Retaining function
Created two his-tagged proteins, one at each end of the DNA sequence encoding for the protein.
Hopefully one or the other will retain function.
Purification of functional protein will allow us to study: ATP binding and hydrolysis, DNA binding, and other functions.

17. Acknowledgements HHMI program, Chris Mathews, Kevin Ahern.
Ream Laboratory; Dr. Walt Ream, Larry Hodges, Jodi Humann, Jen Pitrak.
National Science Foundation
Special thanks to Kevin Ahern for help and support.