Mutations of the alternate start signal for the Galls protein in Agrobacterium rhizogenes

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Mutations of the alternate start signal for the
Galls protein in Agrobacterium rhizogenes

• Henry McNett
• Dr. Walt Ream
• Department of Microbiology

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Agrobacterium tumefaciensNatures Genetic
Engineer
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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

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Agrobacterium tumefaciens plant cell
transformation
Plant cell
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VirE2 and Single-Stranded T-DNA Are Exported
Separately
plant
plant cell
Agrobacterium
nucleus
E1
D2
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Galls replaces VirE2 protein

• Agrobacterium rhizogenes GALLS protein
• Substitutes for A. tumefaciens
  single-stranded DNA-Binding protein VirE2

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

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Genetic representation of the Galls 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.
• How is this other fragment being made?

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Western blot of Galls protein

• Western blot shows presence of shorter C
  Terminus fragment of the protein being made.

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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
• 2) Cleaving of the full length protein
• 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.

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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.

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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.

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The sequence for wild type Galls protein
Ribosome Binding Site
Methionine start codon
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 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'
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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

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Schematic of Methods
Ile
Met
Ligated in PCR product cut with EcoR1 and Pst1
with Methionine to Isoleucine mutation made in
antisense primer
Wild Type Galls plasmid cut with EcoR1 and Pst1

• Transformation into
• Agrobacterium tumefaciens
• for protein analysis

Induced Cre facilitated recombination with
broad host range vector for Agrobacterium
tumefaciens transformation
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EcoRI
Pst I
Pst I
Step 1. Insert EcoRI fragment containing 5 end
of gene.
Step 2. Insert NcoI fragment containing 3 end
of gene.
Pst I
Step 3. Use Crelox site-specific recombination
to insert plasmid containing rebuilt mutant
GALLS gene into a plasmid capable of replicating
in Agrobacterium tumefaciens.
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Results

• Ligated the EcoR1 fragment into plasmid with
  correct orientation
• Ligated the Nco1 fragment into plasmid with
  correct orientation
• Crelox recombination of the plasmid containing
  mutated Galls gene with broad host range vector
• Transform into VirE2 mutant strain of
  Agrobacterium tumefaciens

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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.

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Special thanks

• Dr. Walt Ream
• Howard Hughes Medical Institute
• Larry Hodges
• Dr. Kevin Ahern
• Deborah Moyer