1. Mutations of the alternate start signal for the Galls protein in Agrobacterium rhizogenes Henry McNett Dr. Walt Ream Department of Microbiology

2. Agrobacterium tumefaciens: Nature’s Genetic Engineer

3. Importance of Agrobacterium  Destroys thousands of plants all over the world each year  Is the only bacterium to our knowledge that actually inserts DNA into a eukaryotic organism  Provides very efficient way of transferring DNA into plant cells

4. Agrobacterium tumefaciens plant cell transformation Ti Plasmid T-DNA Agrobacterium Plant cell

5. plant cell E2 D2 E2 Agrobacterium plant E2 D2 E1 E2 E1 nucleu s VirE2 and Single-Stranded T-DNA Are Exported Separately

6. Galls replaces VirE2 protein  Agrobacterium rhizogenes GALLS protein Substitutes for A. tumefaciens single-stranded DNA-Binding protein VirE2 Substitutes for A. tumefaciens single-stranded DNA-Binding protein VirE2

7. A. rhizogenes pRi1724 encodes a protein that substitutes for VirE2 Uninfected control virE2-mutant pTi virE2-mutant pTi + GALLS

8. Galls differs from VirE2  Galls contains an ATP binding motif  A helicase motif  Has three repeats just after the nuclear localization signal  VirE2 is 533 AA whereas Galls is 1769 AA  Both Galls and VirE2 have type 4 secretion signals

9. Genetic representation of the Galls protein GALLS-CT The c terminal part of the protein goes from just after the nuclear localization signal to the c terminal end of the protein.The c terminal part of the protein goes from just after the nuclear localization signal to the c terminal end of the protein. We know this because mutations in the nuclear localization signal do not affect the size of the c terminal fragment.We know this because mutations in the nuclear localization signal do not affect the size of the c terminal fragment. How is this other fragment being made?How is this other fragment being made?

10. Western blot of Galls protein  Western blot shows presence of shorter “C Terminus” fragment of the protein being made.

11. Two possible ways C terminal fragment being made  1) Alternate internal translation start site Translation could be starting internally as well as at the original start codon Translation could be starting internally as well as at the original start codon  2) Cleaving of the full length protein The full length protein could be getting cleaved by the Agrobacterium to serve another function The full length protein could be getting cleaved by the Agrobacterium to serve another function  Hypothesis: The c terminal fragment is made by an internal translational start site. The c terminal fragment is made by an internal translational start site.

12. Project goals  Test the hypothesis that the internal translational start site in the Galls protein is used and is responsible for production of the small c-terminal fragment.

13. Background  Alternate initiation site has 5 out of six base pairs required for an ideal ribosome binding site.  Has methionine as start codon which is required for starting of translation of a protein  Ideal distance between start codon and ribosome binding site 7-9 base pairs, here there are 8.

14. The sequence for wild type Galls protein RBS Start Asp Ser Gly Glu Lys Asn Met Ala Ser 5'-GAC TCA GGA GAA AAA AAT ATG GCT TCG -3' The mutation to the start codon Methionine start codon Methionine replaced by Isoleucine Ribosome Binding Site RBS Asp Ser Gly Glu Lys Asn Ile Ala Ser 5'-GAC TCA GGA GAA AAA AAT ATT GCT TCG -3' Ribosome Binding Site

15. Methods  Alter coding region of Galls to change the methionine coding to isoleucine  Transform into Vir E2-deficient Agrobacterium tumefaciens  Use a western blot to detect the size(s) of Galls being produced by Agrobacterium tumefaciens

16. Schematic of Methods Wild Type Galls plasmid cut with EcoR1 and Pst1 EcoR1Pst1 Ligated in PCR product cut with EcoR1 and Pst1 with Methionine to Isoleucine mutation made in antisense primer EcoR1 Pst1 Induced Cre facilitated recombination with broad host range vector for Agrobacterium tumefaciens transformation Transformation into Agrobacterium tumefaciens for protein analysis Lox site EcoR1Pst1 Met Ile

17. Pst I EcoRI Pst I Step 1. Insert EcoRI fragment containing 5‘ end of gene. Step 2. Insert NcoI fragment containing 3’ end of gene. Step 3. Use Cre:lox site-specific recombination to insert plasmid containing rebuilt mutant GALLS gene into a plasmid capable of replicating in Agrobacterium tumefaciens.

18. Results  Ligated the EcoR1 fragment into plasmid with correct orientation  Ligated the Nco1 fragment into plasmid with correct orientation  Cre:lox recombination of the plasmid containing mutated Galls gene with broad host range vector  Transform into VirE2 mutant strain of Agrobacterium tumefaciens

19. Summary  Galls protein replaces function of VirE2 protein into Agrobacterium tumefaciens  Galls protein is produced in two sizes: full length and a C-terminal fragment that may result from an internal translational start.  Plasmid containing mutation of the putative internal translational start site is built and ready for testing  Virulence assays and western blots are in progress.

20. Special thanks  Dr. Walt Ream  Howard Hughes Medical Institute  Larry Hodges  Dr. Kevin Ahern  Deborah Moyer