Agrobacterium rhizogenes GALLS Protein and Crown Galls

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 • Caused by Agrobacterium tumefaciens transferring oncogenes into plant cells • 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 • 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

4. Background • Agrobacterium tumefaciens contains a tumor-inducing plasmid which encodes oncogenes that cause the plant cell to develop unorganized tumors. A. Tumefaciens infectedbasal surface Uninoculated basal surface

5. Tumor-Inducing (Ti) Plasmid auxin cytokinin octopine ssDNA-binding protein agropine mannopine border endonuclease conjugal plasmid transfer between bacteria overdrive-binding protein Ti Plasmid 200 kb octopine catabolism vir activator mannityl opine catabolism membrane proteins type IV secretion system wound signal receptor/kinase replication origin and incompatibility

6. Interaction Between A. tumefaciens and Plant Cells

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

8. 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

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

13. Putative Nuclear Localization Signal in GALLS Species NucleoproteinNLS Amino Acid Sequence Xenopus laevisnucleoplasminKRPAATKKAGQAKKKKLD A. rhizogenesGALLS (pRi1724)KRKRAAAKEEIDSRKKMARH A. rhizogenes GALLS (pRiA4) KRKRVATKEEIEPHKKMARR A. tumefaciensVirD2 (pTiA6)KRPRDRHDGELGGRKRAR A. tumefaciensVirD2 (pTiC58)KRPREDDDGEPSERKRER A. rhizogenesVirD2 (pRiA4)KRPRVEDDGEPSERKRAR A. tumefaciensVirE2 (pTiA6) #1KLRPEDRYVQTERYGRR A. tumefaciensVirE2 (pTiC58) #1KLRPEDRYIQTEKYGRR A. tumefaciensVirE2 (pTiA6) #2KRRYGGETEIKLKSK A. tumefaciensVirE2 (pTiC58) #2KTKYGSDTEIKLKSK Tobacco Etch Virus NIa proteaseGKKNQKHKLKM(X)31KRKG

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 • Virulence is restored to the VirD2 mutant when a NLS from Tobacco Etch Virus (TEV) is used Wild-type NLS No NLS TEV 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 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 • 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

17. Plasmid jNM 389

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