Transcript Agrobacterium Rhizogeneze

Inducible Expression of an
Agrobacterium rhizogenes Gene
Required for DNA Transfer to Plants
Josh Cuperus, Larry Hodges,
Walt Ream
Department of Microbiology
Oregon State University
Crown Gall


Affects apple, pear, peach, cherry,
almond, raspberry and others.
In Oregon alone causes a million
dollars in damage.
Agrobacterium tumefaciens plant
infection and transformation
Ti plasmid
T-DNA Region
vir gene induction,
single stranded-DNA
released from Ti
plasmid
Agrobacterium attaches to
plant cell and transfers TDNA and virE2, a single
stranded DNA-binding
protein, into plant cell
T-DNA
integrated
into plant
genome
The Tumor Inducing (Ti) plasmid
Wound-Released
Phenolics
periplasm
cytoplasm
VirA
Vir G
Vir G
(inactive)
(active)
VirA
VirB
VirG
VirC VirD
VirE VirF
Plant
Agrobacterium
Nucleus
D2
D2
E2
E1
E2
E2
E1
E2
E1
E2
E2
E2
E2
D2
E2
E2
E2
Agrobacterium rhizogenes



Causes hairy root disease instead of crown
gall.
DNA transfer occurs without virE1 and
virE2 proteins.
Another part of Root- Inducing (Ri)
plasmid encodes a protein that substitutes
for virE2.
Questions



Does Galls protein (ORF 55) have the same
activities as virE2 (ssDNA binding and nuclear
targeting), or does Galls compensate for absence
of virE2?
Is the Galls gene regulated by virA/virG and
phenolic compounds and sugars released by
wounded plant cells?.
Galls protein will help us learn more about gene
transfer to plants.
Galls (ORF 55)
ORF 56
ORF 57 ORF 58
ORF59 ORF 60
ORF 61
Open Reading Frame 55



5.1 kb in length.
virE2 1.6 kb in length and is the largest of
all vir proteins.
Amino acid sequence of ORF 55 is not
related to virE2 region.
Galls protein/ Open reading frame 55
Experimental Approach




Characterize ORF 55 by insertion analysis.
Tn3-LacZ transposon randomly inserted
throughout a cosmid containing ORF 55. lacZ
gene shows if gene is transcribed; ß-galactosidase
(LacZ) converts ONPG (colorless) to onitrophenol (yellow).
lacZ inserted into essential regions will abolish
function and thus tumorigenensis.
Shows whether GALLS gene protein is induced
by acetosyringone, like other vir genes.
lacZ
+
GALLS
cosmid
Transfer into
Agrobacterium
Random insertion
of lacZ throughout
sequence
+
Ti plasmid
Galls
promoter
-AS
Transcriptional fusion (out of frame)
5’ Galls
Lac Z
3’ Galls
DNA
transcription
+AS
RBS
RBS
mRNA
truncated galls protein
Lac Z
= ß galactosidase
Protein
translation
to protein
Galls
promoter
-AS
Translational fusion (In frame)
5’ Galls
Lac Z
3’ Galls
DNA
transcription
+AS
RBS
mRNA
translation
to protein
Partial Galls/ Lac Z
= more
ß galactosidase
Protein
b-galactosidase
activity of GALLS::Tn3-lac insertions
1.8
1.6
1.4
t ranscript ional
f usion
1.2
t ranslat ional
f usion
Units
1
0.8
ant isense1
0.6
ant isense2
0.4
0.2
0
10
20
30
40
t ime (minut es)
50
60
Lac: I
Vir: –
lacZ
I
–
lacZ
I
–
+
lacZ
Galls (ORF 55)
lacZ
Vir:
Lac:
–
+
lacZ
ORF 56
lacZ
ORF 57 ORF 58
lacZ lacZ
–
C
+
lacZ
+
lacZ
ORF59 ORF 60
ORF 61
lacZ lacZ
–
+ +
C C
Vir – Virulence
orientation
I – Inducible
LacZ +
C – Constitutive
LacZ –
Summary



Galls gene is essential for transfer of DNA
into plant cells from Agrobacterium
rhizogenes.
Galls protein substitutes for VirE2.
Galls gene is inducible with
acetosyringone.
Acknowledgements



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HHMI program, Chris Matthews, Kevin Ahern.
Ream Laboratory; Dr. Walt Ream, Larry Hodges, Jodi
Humann, Jen Pitrak.
Buhler laboratory for use of SpectraMax 250.
Special thanks to Kevin Ahern for help and support.