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

Henry McNettHenry McNett

Dr. Walt ReamDr. Walt ReamDepartment of MicrobiologyDepartment of Microbiology

Agrobacterium tumefaciens:Agrobacterium tumefaciens:Nature’s Genetic EngineerNature’s Genetic Engineer

Importance of Agrobacterium Importance of Agrobacterium

Destroys thousands of plants all over the Destroys thousands of plants all over the world each yearworld each year

Is the only bacterium to our knowledge Is the only bacterium to our knowledge that actually inserts DNA into a eukaryotic that actually inserts DNA into a eukaryotic organismorganism

Provides very efficient way of transferring Provides very efficient way of transferring DNA into plant cellsDNA into plant cells

Agrobacterium tumefaciensAgrobacterium tumefaciens plant cell transformationplant cell transformation

Ti Plasmid

T-DNA

Agrobacterium Plant cell

plant cell

E2

D2E2

E2E2

E2

Agrobacterium

plant

E2

D2D2

E1

E1E2

E2

E2

E2

E2

E1

nucleus

VirE2 and Single-Stranded T-DNA Are Exported VirE2 and Single-Stranded T-DNA Are Exported SeparatelySeparately

Galls replaces VirE2 proteinGalls replaces VirE2 protein

Agrobacterium rhizogenesAgrobacterium rhizogenes GALLS protein GALLS protein

Substitutes for Substitutes for A. tumefaciensA. tumefaciens single-stranded single-stranded DNA-Binding protein VirE2DNA-Binding protein VirE2

A. rhizogenes A. rhizogenes pRi1724pRi1724 encodes a protein encodes a protein that substitutes for VirE2that substitutes for VirE2

Uninfected control virE2-mutant pTi virE2-mutant pTi + GALLS

Galls differs from VirE2 Galls differs from VirE2

Galls contains an ATP binding motifGalls contains an ATP binding motif A helicase motifA helicase motif Has three repeats just after the nuclear Has three repeats just after the nuclear

localization signallocalization signal VirE2 is 533 AA whereas Galls is 1769 AAVirE2 is 533 AA whereas Galls is 1769 AA Both Galls and VirE2 have type 4 Both Galls and VirE2 have type 4

secretion signals secretion signals

Genetic representation of the Galls protein

GALLS-CT

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

Western blot of Galls proteinWestern blot of Galls protein

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

Two possible ways C terminal Two possible ways C terminal fragment being madefragment being made

1) Alternate internal translation start site1) Alternate internal translation start site Translation could be starting internally as well as at the original start Translation could be starting internally as well as at the original start

codoncodon

2) Cleaving of the full length protein2) Cleaving of the full length protein The full length protein could be getting cleaved by the Agrobacterium to The full length protein could be getting cleaved by the Agrobacterium to

serve another function serve another function

Hypothesis: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.

Project goalsProject goals

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

BackgroundBackground

Alternate initiation site has 5 out of six base pairs required for an ideal Alternate initiation site has 5 out of six base pairs required for an ideal ribosome binding site. ribosome binding site.

Has methionine as start codon which is required for starting of translation of a Has methionine as start codon which is required for starting of translation of a proteinprotein

Ideal distance between start codon and ribosome binding site 7-9 base pairs, Ideal distance between start codon and ribosome binding site 7-9 base pairs, here there are 8.here there are 8.

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

MethodsMethods

Alter coding region of Galls to change the Alter coding region of Galls to change the methionine coding to isoleucine methionine coding to isoleucine

Transform into Vir E2-deficient Transform into Vir E2-deficient Agrobacterium tumefaciensAgrobacterium tumefaciens

Use a western blot to detect the size(s) of Use a western blot to detect the size(s) of Galls being produced by Galls being produced by Agrobacterium Agrobacterium tumefacienstumefaciens

Schematic of Methods

Wild Type Galls plasmid cut with EcoR1 and Pst1

EcoR1 Pst1

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 Agrobacterium tumefaciens transformationtransformation

Transformation into

Agrobacterium tumefaciensAgrobacterium tumefaciens for protein analysis

Lox site

Lox site

EcoR1 Pst1

Met

Ile

Pst IPst IEcoRI

Pst IStep 1. Insert EcoRI fragment containing5‘ end of gene.

Step 2. Insert NcoIfragment containing3’ 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.

ResultsResults

Ligated the Ligated the EcoEcoR1 fragment into plasmid R1 fragment into plasmid with correct orientationwith correct orientation

Ligated the Ligated the NcoNco1 fragment into plasmid 1 fragment into plasmid with correct orientationwith correct orientation

Cre:lox recombination of the plasmid Cre:lox recombination of the plasmid containing mutated containing mutated Galls Galls gene with broad gene with broad host range vectorhost range vector

Transform into VirE2 mutant strain of Transform into VirE2 mutant strain of Agrobacterium tumefaciensAgrobacterium tumefaciens

SummarySummary

Galls protein replaces function of VirE2 protein Galls protein replaces function of VirE2 protein into into Agrobacterium tumefaciensAgrobacterium tumefaciens

Galls protein is produced in two sizes: full length Galls protein is produced in two sizes: full length and a C-terminal fragment that may result from and a C-terminal fragment that may result from an internal translational start.an internal translational start.

Plasmid containing mutation of the putative Plasmid containing mutation of the putative internal translational start site is built and ready internal translational start site is built and ready for testingfor testing

Virulence assays and western blots are in Virulence assays and western blots are in progress.progress.

Special thanks Special thanks

Dr. Walt ReamDr. Walt Ream Howard Hughes Medical Institute Howard Hughes Medical Institute Larry Hodges Larry Hodges Dr. Kevin AhernDr. Kevin Ahern Deborah MoyerDeborah Moyer