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Inducible Expression of an Agrobacterium rhizogenes Gene Required for DNA Transfer to Plants

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. Crown Gall. Affects apple, pear, peach, cherry, almond, raspberry and others.

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Inducible Expression of an Agrobacterium rhizogenes Gene Required for DNA Transfer to Plants

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  1. Inducible Expression of an Agrobacterium rhizogenes GeneRequired 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. Agrobacteriumtumefaciens plant infection and transformation Ti plasmid T-DNA Region vir gene induction, single stranded-DNA released from Ti plasmid T-DNA integrated into plant genome Agrobacterium attaches to plant cell and transfers T-DNA and virE2, a single stranded DNA-binding protein, into plant cell

  4. The Tumor Inducing (Ti) plasmid

  5. Wound-Released Phenolics periplasm cytoplasm VirA Vir G (inactive) Vir G (active) VirD VirA VirB VirG VirC VirE VirF

  6. Plant Agrobacterium Nucleus E2 D2 E2 D2 E2 E2 E2 E2 E1 D2 E2 E2 E1 E2 E2 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 58 ORF 56 ORF 57 ORF59 ORF 60 ORF 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. lac lac lacZ + + Random insertion of lacZ throughout sequence GALLS cosmid lac Ti plasmid Transfer into Agrobacterium

  14. Transcriptional fusion (out of frame) Galls promoter -AS 5’ Galls Lac Z 3’ Galls DNA transcription +AS RBS RBS mRNA truncated galls protein translation to protein = ß galactosidase Protein Lac Z

  15. Galls promoter Translational fusion (In frame) -AS 5’ Galls Lac Z 3’ Galls DNA transcription +AS RBS mRNA translation to protein Lac Z Partial Galls/ = more ß galactosidase Protein

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

  17. Lac: I I I C Vir: – – – + + + + lacZ lacZ lacZ lacZ lacZ lacZ lacZ Galls (ORF 55) ORF 58 ORF 56 ORF 57 ORF59 ORF 60 ORF 61 lacZ lacZ lacZ lacZ lacZ Vir: – – – + + 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.

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