Transcript Agrobacterium Gene Transfer to Plants rhizogenes Josh Cuperus, Larry Hodges,

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
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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
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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
Agrobacterium
Nucleus
D2
D2
E2
E1
E2
E2
E1
E2
E1
E2
E2
E2
E2
D2
E2
E2
E2
Agrobacterium rhizogenes
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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
plant
nucleu
s
VirB/D4
D2
D2
Agrobacterium rhizogenes
D2
GALLS
VirB/D4
GALLS
Domains in the GALLS Protein
NTP-Binding
TraA-Like
T4SS
Similarities between GALLS and VirE2
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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
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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
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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
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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
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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
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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.