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

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)

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

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

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

Interaction Between Interaction Between A. tumefaciensA. tumefaciens and Plant Cellsand Plant Cells

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

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

Arrangement of Virulence Operons in Ti & Arrangement of Virulence Operons in Ti & Ri PlasmidsRi Plasmids

Ri & Ti Plasmid MapsRi & Ti Plasmid Maps

A. rhizogenes A. rhizogenes pRi1724pRi1724 Galls protein Galls protein substitutes for VirE2substitutes for VirE2

virE2-mutant pTi + pRi1724 virE2-mutant pTi

Domains in the GALLS Protein

NTP-Binding TraA-Like T4SS

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

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

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

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

Plasmid jNM 389

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