1. Inducible Expression of an Agrobacterium rhizogenes Gene Required for DNA Transfer to Plants Josh Cuperus, Larry Hodges, Walt Ream Department of Microbiology Oregon State University

2. Crown Gall Affects apple, pear, peach, cherry, almond, raspberry and others. In Oregon alone causes a million dollars in damage.

3. Agrobacterium tumefaciens plant infection and transformation Ti plasmidT-DNA Region vir gene induction, single stranded-DNA released from Ti plasmid Agrobacterium attaches to plant cell and transfers T- DNA and virE2, a single stranded DNA-binding protein, into plant cell T-DNA integrated into plant genome

4. The Tumor Inducing (Ti) plasmid

5. VirA cytoplasm periplasm Vir G (inactive) Vir G (active) VirAVirBVirGVirC VirD VirEVirF Wound-Released Phenolics

6. Plant D2 E2 D2 E2 Nucleus E2 Agrobacterium E2 D2 E1 E2 E1

7. Agrobacterium rhizogenes Causes hairy root disease instead of crown gall. DNA transfer occurs without virE1 and virE2 proteins. Another part of Root- Inducing (Ri) plasmid encodes a protein that substitutes for virE2.

8. Questions Does Galls protein (ORF 55) have the same activities as virE2 (ssDNA binding and nuclear targeting), or does Galls compensate for absence of virE2? Is the Galls gene regulated by virA/virG and phenolic compounds and sugars released by wounded plant cells?. Galls protein will help us learn more about gene transfer to plants.

9. Galls (ORF 55) ORF 56ORF 57 ORF 58 ORF59ORF 60ORF 61

10. Open Reading Frame 55 5.1 kb in length. virE2 1.6 kb in length and is the largest of all vir proteins. Amino acid sequence of ORF 55 is not related to virE2 region.

11. Galls protein/ Open reading frame 55

12. Experimental Approach Characterize ORF 55 by insertion analysis. Tn3-LacZ transposon randomly inserted throughout a cosmid containing ORF 55. lacZ gene shows if gene is transcribed; ß-galactosidase (LacZ) converts ONPG (colorless) to o- nitrophenol (yellow). lacZ inserted into essential regions will abolish function and thus tumorigenensis. Shows whether GALLS gene protein is induced by acetosyringone, like other vir genes.

13. lacZ + GALLS cosmid lac Random insertion of lacZ throughout sequence + lac Transfer into Agrobacterium lac Ti plasmid

14. Galls promoter -AS 5’ GallsLac Z3’ Galls +AS DNA transcription mRNA RBS translation to protein truncated galls protein Lac Z = ß galactosidase Protein Transcriptional fusion (out of frame)

15. Galls promoter -AS 5’ GallsLac Z3’ Galls +AS DNA transcription mRNA RBS translation to protein Lac Z = more ß galactosidase Protein Partial Galls/ Translational fusion (In frame)

16. Units  galactosidase activity of GALLS::Tn3-lac insertions

17. Galls (ORF 55) ORF 56ORF 57 ORF 58 ORF59ORF 60ORF 61 lacZ Vir: – – – + + + + Vir: – – – + + Lac: I I I C Lac: C C Vir – Virulence I – Inducible C – Constitutive orientation LacZ + LacZ –

18. Summary Galls gene is essential for transfer of DNA into plant cells from Agrobacterium rhizogenes. Galls protein substitutes for VirE2. Galls gene is inducible with acetosyringone.

19. Acknowledgements HHMI program, Chris Matthews, Kevin Ahern. Ream Laboratory; Dr. Walt Ream, Larry Hodges, Jodi Humann, Jen Pitrak. Buhler laboratory for use of SpectraMax 250. Special thanks to Kevin Ahern for help and support.