Transcript Agrobacterium rhizogenes GALLS Protein and Crown Galls

Agrobacterium rhizogenes
GALLS Protein and Crown
Galls
Jason Neal-McKinney
Dr. Walt Ream
Department of Microbiology
Crown Gall Disease
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Formation of unorganized tumors on
plants, especially the roots
Caused by Agrobacterium tumefaciens
transferring oncogenes into plant cells
Crown Galls affect almost every plant
species (except monocots)
Importance of Crown Gall
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Crown gall tumors interfere with water and
nutrient flow, resulting in stunted or nonproductive plants
Economically, it is a major problem for farmers
and nurseries
The bacteria which cause crown gall disease can
be used to transfer beneficial DNA into plants as
well
Background
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Agrobacterium tumefaciens contains a
tumor-inducing plasmid which encodes
oncogenes that cause the plant cell to
develop unorganized tumors.
A. Tumefaciens infected
basal surface
Uninoculated basal
surface
Tumor-Inducing (Ti) Plasmid
auxin
cytokinin
octopine
ssDNA-binding
protein
agropine
mannopine
border
endonuclease
conjugal plasmid
transfer between
bacteria
overdrivebinding protein
Ti Plasmid
200 kb
vir activator
octopine
catabolism
mannityl
opine
catabolism
membrane proteins
type IV secretion system
wound signal
receptor/kinase
replication origin
and incompatibility
Interaction Between A. tumefaciens and
Plant Cells
VirE2 and Single-Stranded T-DNA Are Exported Separately
Plant Cellplant cell
Nucleus
Agrobacterium Cell
E2
E2
E2
E2
D2
D2
E2
E1
E2
E2
E1
E2
E1
D2
E2
E2
E2
Agrobacterium rhizogenes Causes Hairy Root Disease;
A. tumefaciens Causes Unorganized Tumors (Crown
Galls)
A. tumefaciens
basal surface
A. rhizogenes
apical surface
uninoculated
basal surface
uninoculated
apical surface
Arrangement of Virulence Operons in Ti & Ri
Plasmids
Ri & Ti Plasmid Maps
A. rhizogenes pRi1724 Galls protein
substitutes for VirE2
virE2-mutant pTi + pRi1724
virE2-mutant pTi
Domains in the GALLS Protein
NTP-Binding
TraA-Like
T4SS
Putative Nuclear Localization Signal in
GALLS
Species
Xenopus laevis
Nucleoprotein
nucleoplasmin
NLS Amino Acid Sequence
KRPAATKKAGQAKKKKLD
A. rhizogenes
A. rhizogenes
GALLS (pRi1724)
GALLS (pRiA4)
KRKRAAAKEEIDSRKKMARH
KRKRVATKEEIEPHKKMARR
A. tumefaciens
A. tumefaciens
A. rhizogenes
VirD2 (pTiA6)
VirD2 (pTiC58)
VirD2 (pRiA4)
KRPRDRHDGELGGRKRAR
KRPREDDDGEPSERKRER
KRPRVEDDGEPSERKRAR
A. tumefaciens
A. tumefaciens
A. tumefaciens
A. tumefaciens
VirE2 (pTiA6) #1
VirE2 (pTiC58) #1
VirE2 (pTiA6) #2
VirE2 (pTiC58) #2
KLRPEDRYVQTERYGRR
KLRPEDRYIQTEKYGRR
KRRYGGETEIKLKSK
KTKYGSDTEIKLKSK
Tobacco Etch Virus
NIa protease
GKKNQKHKLKM(X)3 1KRKG
The NLS in VirD2 is necessary
for tumor formation
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When the NLS is removed from wild-type
VirD2, T-DNA production continues,
although no tumors are formed
Virulence is restored to the VirD2 mutant
when a NLS from Tobacco Etch Virus
(TEV) is used
Wild-type NLS
No NLS
TEV NLS
Project Goals
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To compare the efficiency of DNA transfer
between Agrobacterium strains containing the
Galls gene with the wild-type NLS, no NLS, and
an NLS from Tobacco Etch Virus, which is very
different from the wild-type NLS
To determine whether the NLS is necessary for
the Galls protein to function and interact with
host cell proteins
Experiment Layout
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Step 1: Create 3 plasmids containing the
different versions of the GALLS gene
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pJNM 389- Wild Type NLS
pJNM 390- No NLS
pJNM 392- Tobacco Etch Virus NLS
Step 2: Transform the 3 plasmids into a
Agrobacteria tumefaciens strain lacking
the VirE2 gene
Step 3: Inoculate carrot surfaces with the
3 different strains and observe tumor
formation
Plasmid jNM 389
Special Thanks to:
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Dr. Walt Ream
The National Science Foundation
Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. Larry Hodges
Dr. Jodi Humann