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

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 Bacterial Cell Plant Cell T-DNA integrated into plant genome

Plant D2 E2 Nucleus Agrobacterium 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

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

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.