purification of a secreted agrobacterium rhizogenes protein(galls) required for gene transfer to...

Purification of a Secreted Agrobacterium rhizogenes Protein(GALLS) Required for

Gene Transfer to Plants

Josh Cuperus, Larry Hodges, Dr. Walt Ream

Department of MicrobiologyOregon State University

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

Crown Gall Disease

Caused by Agrobacterium tumefaciens

Agrobacterium tumefaciens plant infection and transformation

Ti plasmid T-DNA Region

T-DNA integrated into plant genome

Bacterial Cell

Plant Cell

Plant

D2E2

E2E2

E2

D2

E2

E2

E2

E2

Nucleus

E2

Agrobacterium

E2

D2

E1

E1

E2

E1

Agrobacterium rhizogenes Causes hairy root disease instead of crown

gall. DNA transfer occurs without two essential

proteins (VirE1 and VirE2) found in Agrobacterium tumefaciens.

Arrangement of Virulence Operons in Ti & Ri Plasmids

Ri & Ti Plasmid Maps

Ti Plasmid Ri Plasmid

D2

plantA. tumefaciens

D2 D2

nucleus

GALLS Replaces VirE2 by Mixed Infection

Agrobacterium rhizogenes

GALLSGALLSVirB/D4

VirB/D4

Domains in the GALLS Protein

NTP-Binding TraA-Like T4SS

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.

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.

Purpose Protein purification Use in creating antibodies that will work on

normal GALLS protein Antibodies will allow recognition of protein

in other studies

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.

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.

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.