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Agrobacterium rhizogenesAgrobacterium rhizogenes GALLS Protein and Crown GALLS Protein and Crown
GallsGalls
Jason Neal-McKinneyJason Neal-McKinneyDr. Walt ReamDr. Walt Ream
Department of MicrobiologyDepartment of Microbiology
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Crown Gall DiseaseCrown Gall Disease Formation of unorganized tumors on Formation of unorganized tumors on
plants, especially the rootsplants, especially the roots Caused by Caused by Agrobacterium Agrobacterium
tumefaciens tumefaciens transferring oncogenes transferring oncogenes into plant cellsinto plant cells
Crown Galls affect almost every plant Crown Galls affect almost every plant species (except monocots)species (except monocots)
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Importance of Crown GallImportance of Crown Gall Crown gall tumors interfere with water and Crown gall tumors interfere with water and
nutrient flow, resulting in stunted or non-nutrient flow, resulting in stunted or non-productive plantsproductive plants
Economically, it is a major problem for Economically, it is a major problem for farmers and nurseriesfarmers and nurseries
The bacteria which cause crown gall The bacteria which cause crown gall disease can be used to transfer beneficial disease can be used to transfer beneficial DNA into plants as wellDNA into plants as well
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BackgroundBackground Agrobacterium tumefaciens Agrobacterium tumefaciens contains contains
a tumor-inducing plasmid which a tumor-inducing plasmid which encodes oncogenes that cause the encodes oncogenes that cause the plant cell to develop unorganized plant cell to develop unorganized tumors.tumors.
A. Tumefaciens infected basal surface
Uninoculated basal surface
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Ti Plasmid
200 kb
auxin
cytokinin
octopine
mannityl opine
catabolism
octopine catabolism
vir activator
overdrive-binding protein
border endonuclease
ssDNA-binding protein agropine
mannopine
conjugal plasmid transfer between
bacteria
membrane proteins
type IV secretion system
wound signal receptor/kinase
replication origin and
incompatibility
Tumor-Inducing (Ti) Plasmid
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Interaction Between Interaction Between A. tumefaciensA. tumefaciens and Plant Cellsand Plant Cells
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plant cell
E2
D2E2
E2E2
E2
Agrobacterium Cell
Plant Cell
E2
D2D2
E1
E1E2
E2
E2
E2
E2
E1
Nucleus
VirE2 and Single-Stranded T-DNA Are Exported Separately
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Agrobacterium rhizogenesAgrobacterium rhizogenes Causes Hairy Root Causes Hairy Root Disease;Disease;
A. tumefaciens A. tumefaciens Causes Unorganized Tumors Causes Unorganized Tumors (Crown Galls)(Crown Galls)
A. tumefaciensbasal surface
uninoculatedbasal surface
A. rhizogenesapical surface
uninoculatedapical surface
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Arrangement of Virulence Operons in Ti & Arrangement of Virulence Operons in Ti & Ri PlasmidsRi Plasmids
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Ri & Ti Plasmid MapsRi & Ti Plasmid Maps
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A. rhizogenes A. rhizogenes pRi1724pRi1724 Galls protein Galls protein substitutes for VirE2substitutes for VirE2
virE2-mutant pTi + pRi1724 virE2-mutant pTi
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Domains in the GALLS Protein
NTP-Binding TraA-Like T4SS
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Putative Nuclear Localization Signal Putative Nuclear Localization Signal in GALLSin GALLS
Species Nucleoprotein NLS Amino Acid SequenceXenopus laevis nucleoplasmin KRPAATKKAGQAKKKKLD A. rhizogenes GALLS (pRi1724)
KRKRAAAKEEIDSRKKMARHA. rhizogenes GALLS (pRiA4)
KRKRVATKEEIEPHKKMARR
A. tumefaciens VirD2 (pTiA6) KRPRDRHDGELGGRKRARA. tumefaciens VirD2 (pTiC58) KRPREDDDGEPSERKRERA. rhizogenes VirD2 (pRiA4) KRPRVEDDGEPSERKRAR A. tumefaciens VirE2 (pTiA6) #1 KLRPEDRYVQTERYGRRA. tumefaciens VirE2 (pTiC58) #1 KLRPEDRYIQTEKYGRRA. tumefaciens VirE2 (pTiA6) #2 KRRYGGETEIKLKSKA. tumefaciens VirE2 (pTiC58) #2 KTKYGSDTEIKLKSK Tobacco Etch Virus NIa protease GKKNQKHKLKM(X)3 1KRKG
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The NLS in VirD2 is necessary The NLS in VirD2 is necessary for tumor formationfor tumor formation
When the NLS is removed from wild-When the NLS is removed from wild-type VirD2, T-DNA production type VirD2, T-DNA production continues, although no tumors are continues, although no tumors are formedformed
Virulence is restored to the VirD2 Virulence is restored to the VirD2 mutant when a NLS from Tobacco mutant when a NLS from Tobacco Etch Virus (TEV) is usedEtch Virus (TEV) is used
Wild-type NLS No NLS TEV NLS
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Project GoalsProject Goals To compare the efficiency of DNA transfer To compare the efficiency of DNA transfer
between between AgrobacteriumAgrobacterium strains containing strains containing the Galls gene with the wild-type NLS, no the Galls gene with the wild-type NLS, no NLS, and an NLS from Tobacco Etch Virus, NLS, and an NLS from Tobacco Etch Virus, which is very different from the wild-type NLSwhich is very different from the wild-type NLS
To determine whether the NLS is necessary To determine whether the NLS is necessary for the Galls protein to function and interact for the Galls protein to function and interact with host cell proteinswith host cell proteins
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Experiment LayoutExperiment Layout Step 1: Create 3 plasmids containing Step 1: Create 3 plasmids containing
the different versions of the GALLS the different versions of the GALLS genegene pJNM 389- Wild Type NLSpJNM 389- Wild Type NLS pJNM 390- No NLSpJNM 390- No NLS pJNM 392- Tobacco Etch Virus NLSpJNM 392- Tobacco Etch Virus NLS
Step 2: Transform the 3 plasmids into a Step 2: Transform the 3 plasmids into a Agrobacteria tumefaciensAgrobacteria tumefaciens strain strain lacking the VirE2 genelacking the VirE2 gene
Step 3: Inoculate carrot surfaces with Step 3: Inoculate carrot surfaces with the 3 different strains and observe the 3 different strains and observe tumor formationtumor formation
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Plasmid jNM 389
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Special Thanks to:Special Thanks to:
Dr. Walt ReamDr. Walt Ream The National Science Foundation The National Science Foundation Howard Hughes Medical InstituteHoward Hughes Medical Institute Dr. Kevin AhernDr. Kevin Ahern Dr. Larry HodgesDr. Larry Hodges Dr. Jodi HumannDr. Jodi Humann