1. Agrobacterium rhizogenes GALLS Protein and Crown Galls Jason Neal-McKinney Dr. Walt Ream Department of Microbiology

2. Crown Gall Disease Formation of unorganized tumors on plants, especially the roots Formation of unorganized tumors on plants, especially the roots Caused by Agrobacterium tumefaciens transferring oncogenes into plant cells Caused by Agrobacterium tumefaciens transferring oncogenes into plant cells Crown Galls affect almost every plant species (except monocots) Crown Galls affect almost every plant species (except monocots)

3. Importance of Crown Gall Crown gall tumors interfere with water and nutrient flow, resulting in stunted or non- productive plants Crown gall tumors interfere with water and nutrient flow, resulting in stunted or non- productive plants Economically, it is a major problem for farmers and nurseries 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 The bacteria which cause crown gall disease can be used to transfer beneficial DNA into plants as well

4. Background Agrobacterium tumefaciens contains a tumor-inducing plasmid which encodes oncogenes that cause the plant cell to develop unorganized tumors. 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

5. 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

6. Interaction Between A. tumefaciens and Plant Cells

7. plant cell E2 D2 E2 Agrobacterium Cell Plant Cell E2 D2 E1 E2 E1 Nucleus VirE2 and Single-Stranded T-DNA Are Exported Separately

8. Agrobacterium rhizogenes Causes Hairy Root Disease; A. tumefaciens Causes Unorganized Tumors (Crown Galls) A. tumefaciens basal surface uninoculated basal surface A. rhizogenes apical surface uninoculated apical surface

9. Arrangement of Virulence Operons in Ti & Ri Plasmids

10. Ri & Ti Plasmid Maps

11. A. rhizogenes pRi1724 Galls protein substitutes for VirE2 virE2-mutant pTi + pRi1724 virE2-mutant pTi

12. Domains in the GALLS Protein NTP-Binding TraA-Like T4SS

13. Putative Nuclear Localization Signal in GALLS Species NucleoproteinNLS Amino Acid Sequence Xenopus laevis nucleoplasmin KRPAATKKAGQAKKKKLD A. rhizogenesGALLS (pRi1724) KRKRAAAKEEIDSRKKMARH A. rhizogenesGALLS (pRiA4) KRKRVATKEEIEPHKKMARR A. tumefaciensVirD2 (pTiA6) KRPRDRHDGELGGRKRAR A. tumefaciensVirD2 (pTiC58) KRPREDDDGEPSERKRER A. rhizogenesVirD2 (pRiA4) KRPRVEDDGEPSERKRAR A. tumefaciensVirE2 (pTiA6) #1 KLRPEDRYVQTERYGRR A. tumefaciensVirE2 (pTiC58) #1 KLRPEDRYIQTEKYGRR A. tumefaciensVirE2 (pTiA6) #2 KRRYGGETEIKLKSK A. tumefaciensVirE2 (pTiC58) #2 KTKYGSDTEIKLKSK Tobacco Etch VirusNIa protease GKKNQKHKLKM (X) 3 1 KRKG

14. The NLS in VirD2 is necessary for tumor formation When the NLS is removed from wild-type VirD2, T-DNA production continues, although no tumors are formed 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 Virulence is restored to the VirD2 mutant when a NLS from Tobacco Etch Virus (TEV) is used Wild-type NLSNo NLSTEV NLS

15. Project Goals 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 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 To determine whether the NLS is necessary for the Galls protein to function and interact with host cell proteins

16. Experiment Layout Step 1: Create 3 plasmids containing the different versions of the GALLS gene Step 1: Create 3 plasmids containing the different versions of the GALLS gene pJNM 389- Wild Type NLS pJNM 389- Wild Type NLS pJNM 390- No NLS pJNM 390- No NLS pJNM 392- Tobacco Etch Virus NLS pJNM 392- Tobacco Etch Virus NLS Step 2: Transform the 3 plasmids into a Agrobacteria tumefaciens strain lacking the VirE2 gene 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 Step 3: Inoculate carrot surfaces with the 3 different strains and observe tumor formation

17. Plasmid jNM 389

18. Special Thanks to: Dr. Walt Ream Dr. Walt Ream The National Science Foundation The National Science Foundation Howard Hughes Medical Institute Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Kevin Ahern Dr. Larry Hodges Dr. Larry Hodges Dr. Jodi Humann Dr. Jodi Humann