Understanding expression of
Agrobacterium rhizogenes proteins
(GALLS) required for gene transfer to plants.
By
Chris Brown
Dr. Walt Ream’s Laboratory
Microbiology department

Background
•
Agrobacterium damages at least 1.4 million dollars worth of agriculture per year in
California and Oregon alone.

•
Agrobacterium is the only known prokaryote to transfer genes to a eukaryote.
•
Agrobacterium is used throughout microbiology to transfer DNA into plant cells
Agrobacterium tumefaciens attached to a plant cell.
Image by Martha Hawes

•
Agrobacterium tumefaciens and A. rhizogenes infect wounded plants and transfer plasmid DNA
(T-DNA) and virulence (Vir) proteins into plant cells. http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg
http://www.nature.com/nature/jour nal/v433/n7026/images/433583af2.2.jpg

Understanding the GALLS proteins
Fig. 1.2 GALLS
GALLS Full Length
Repeat 1
GALLS-FL
Repeat 2
GALLS-CT
Repeat 3
GALLS C Terminal

Research Question:
What causes the lower quantity of GALLS-FL relative to GALLS-CT ?
GALLS
Hypothesis:
Two Causes
1. mRNA degradation
2. Codon bias
FL
170
130
CT
100

GALLS-FL probes
C
A
GALLS-CT probes
D
B
Repeat 1 Repeat 2 Repeat 3
Sense probe
Antisense probe
Ratio of Sense RNA levels
Average
Ratio A to B
1.04 +0.37
Ratio of Antisense to Sense RNA levels
C to A .081
+.0003
D to B .106
+.009

Facts about Codon Bias
• Codon UUA is found in .075% of leucines in
GALLS
• CUA is found in 5% of leucines in
GALLS
• Most common leucine in
GALLS is CUC
•
GALLS contains 8 of these rare codons
• 7 CUA 1 UUA
• GALLS-FL upstream of GALLS-CT contains
6 of the 8 rare codons including the single
UUA

Fig. 1.2
Location of rare leucine codons
UUA (152 codon)
CUA (606 codon)
CUA (495 codon)
CUA (740 codon)
CUA (1347 codon)
CUA (1139 codon)
Repeat 1
GALLS-FL
Repeat 2
GALLS-CT
Repeat 3
CUA (83 codon) CUA (254 codon)

Method for Overcoming Codon Bias
Over expression of tRNA gene corresponding to
CUA
• Located and amplified tRNA gene from
Agrobacterium rhizogenes genome using
Polymerase Chain Reaction (PCR)
• Inserted amplicon of tRNA corresponding to leucine CUA into pCR4-TOPO® vector
• Inserted loxP site into plasmid
PCR product loxP site pCR4-TOPO®

Method for Overcoming Codon Bias cont.
• Recombined the plasmid containing rare leucine tRNA gene corresponding to CUA with broad-host-range plasmid (compatible with
•
A. tumefaciens ) via Cre-Lox recombination

Cre-Lox
5`CATACA 3 `
3`GTATGT 5`
3`GTATGT 5`
5`CATACA 3 `
3`GTATGT 5`
5`CATACA 3`
3`GTATGT 5`
5`CATACA 3`
3`ACATAC 5`
5`TGTATG 3`
3`GTATGT 5`
5`TGTATG 3`

• Recombined the plasmid containing rare leucine tRNA gene corresponding to CUA with broad host range plasmid (compatible with A. tumefaciens ) via Cre-Lox recombination
Cre protein Mediated pCR4-TOPO vector tRNA gene (CUA) loxP site

Method for Overcoming Codon Bias Cont.
• Transformed cointegrate (broad-hostrange/tRNA containing plasmid) into A. tumafaciens
• Expressed gene and determine protein yield

Method for overcoming Codon Bias
Part 2
Replace rarest leucine codon (UUA) with most common leucine codon (CUC)
•Create mutation in TTA (UUA) codon 152 of
Galls from Leu UUA to Leu CUC using overlap extension Polymerase Chain reaction
UUA (154 codon)
GALLS-CT
PCR product

5`
3`
A
1. RXN with primers A & C
A
5`
3`
B
C
2. Combination of
DNA from Reactions in step one with out primers (self priming)
5`
3`
C
B
3`
5`
5` 3`
1. RXN with primers B & D
5`
RXN with Primers A & D
5`
3`
A
D
D
3`
5`
5`
D
3`
5`
5`

Method for overcoming Codon Bias
Part 2C
• Insert PCR product with leucine mutation into vector containing the flanking sequence of Galls
Vector
Vector
ΔUUA (154 codon)
Galls
Flanking
PCR product
Galls
Flanking
PCR product
GALLS-CT

Method for overcoming Codon Bias
Part 2C
• Insert entire plasmid containing
Galls -Δleu154
(UUA CUC) into an A. tumafaciens compatible vector
• Transform into
A. tumafaciens

Results of tRNA over expression
+ tRNA
FL
170 kD
130 kD
CT
CUA (1347 codon)
CUA (1139 codon)
CUA (606 codon)
CUA (495 codon)
CUA (740 codon)
Repeat
1
GALLS-FL
Repeat Repeat
3
CUA (83 codon) CUA (254 codon)

Results of Codon 152
(Leu UUA to CUC)
Mutation
• Transformation of DNA unsuccessful
• Co-transformation yielded all colonies containing only plasmid the not containing
GALLS
• Possible toxicity to
A. rhizogenes

Future Work
• Use a regulated non-
Agrobacterium promoter to allow control of GALLS-FL production

• HHMI
• COS Cripps Scholarship Fund
• Dr. Kevin Ahern
• Larry Hodges
• Dr. Walt Ream