Purification, Crystallization, and
Mutagenesis of ToxB, a HostSelective Toxin in Pyrenophora
tritici-repentis
Kara Miles-Rockenfield
In collaboration with the labs of
Drs. Lynda Ciuffetti1 and P. Andrew Karplus2
1Department
of Botany and Plant Pathology
2Department
of Biochemistry and Biophysics
Tan Spot




One of the major diseases
affecting wheat
Wide geographical
distribution
Causes lightweight and
shriveled grains
Results in 3-50% crop loss
(Strelkov and Lamari 2003)
…and we know what causes it!
Pyrenophora tritici-repentis


Fungus that causes
tan-spot in wheat
Symptoms caused
by Host-Selective
Toxins

Molecules
secreted by the
fungus
Host-Selective Toxins

Produced by fungi
 Small
molecular weight molecules
 Proteins

Reproduce symptoms of disease
 Pathogen
not required
Primary determinants of pathogenicity
 Toxic only to susceptible plants

 Non-host
plants not affected
Symptoms of HSTs

Necrosis


Chlorosis

Healthy Leaf
Chlorosis
Cell death
Breakdown of chlorophyll
Necrotic Lesions
HST of Ptr

ToxB
Protein
 Causes chlorosis
 Multiple copies of the gene cause greater
chlorosis
 Inactive form toxb

Zone of Infiltration
Hypothesis
A specific region of the structure of
ToxB is responsible for the toxicity
of the protein.
Project Goals

Purify ToxB from Ptr

Mutagenesis
 Important regions
of the gene for
toxicity
First Goal
Purification of ToxB
Purification of ToxB
Protein Production in Crude Culture Filtrate
Plugs vs. Ground
Harvest Filtrate
Filters
Protein Precipitations
2-Step (NH4)2SO4
Chromatography
Cation Exchange
Fraction Analysis
Silver Stain, Western Blot, Protein Assay
Purification Results

Western Blot
Western Blot
 Silver Stain
 BCA Protein
Assay

kD
P
F1 F2 W1 W2 W3 M E1
E2 E3 E4
75
50
Silver Stain
Protein Analysis
37
25
20

15
10
ToxB
Additional
purification needed
Second Goal
Mutagenesis of ToxB
Sequence Comparison
I
II
III
toxb MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC
ToxB MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC

Differences between the
sequences
 Deletion in ToxB
 Proline in toxb

Restriction sites present in both
 Divides protein into three regions
H2
C
H2C
N
CH2
C
H
H
COOH
Chimeras
 Chimeric
Proteins
 Swap Coding Regions
pCMR3 – BBb
I
II
III
MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC
pCMR4 – bbB
MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC
pCMR5 – Bbb
MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC
pCMR6 – bBB
MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC
pCMR7 – BbB
MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC
pCMR8 – bBb
MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC
ToxB/toxb Plasmids

ampicillin
Subcloned into pBSII

pCMR1-toxb
Bsa I (2417)
3270 bp
Eco RI (702)
I
II
III
Bse YI (845)
One site each of
BseYI and BsaI
toxb
Bsa I (924)
Not I (1048)
ampicillin
pUC ori
pCMR2-ToxB
Bsa I (2414)
3267 bp
Bse YI (1767)
Eco RI (702)
I
II
III
Bse YI (845)
ToxB
Bsa I (924)
Not I (1045)
pUC ori
Bse YI (1764)
Expression of Chimeras

Subcloning into expression vector
 pPICZB

Transformation into Pichia pastoris
 Expression
of chimeras
 Secretes protein
Summary of Purification

Partial purification of ToxB
Next Steps of Purification
Finish purification
 Crystallization of ToxB
 3-D Structure

Summary of Mutagenesis

BBb

BBb
Generation of 5 of 6 chimeras
bbB
Bbb
bBB
BbB
bBb
Two chimeras transformed into Pichia
bbB
Bbb
bBB
BbB
bBb
Next Steps of Mutagenesis
 Finish
generation of chimeras
 Transform into Pichia
 Bioassay
 Additional chimeras?
Acknowledgements
HHMI
 URISC
 Dr. Lynda Ciuffetti
 Dr. P. Andrew Karplus
 Viola Manning
 Dr. Iovanna Pandelova
 Dr. Kevin Ahern
