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Kara Miles-RockenfieldIn collaboration with the labs of
Drs. Lynda Ciuffetti1 and P. Andrew Karplus2
Purification, Crystallization, and
Mutagenesis of ToxB, a Host-Selective Toxin in
Pyrenophora tritici-repentis
1Department of Botany and Plant Pathology
2Department of Biochemistry and Biophysics
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Tan Spot
One of the major diseases affecting wheat
Wide geographical distribution
Causes lightweight and shriveled grains
Results in 3-50% crop loss
…and we know what causes it!
(Strelkov and Lamari 2003)
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Pyrenophora tritici-repentis
Fungus that causes tan-spot in wheat
Symptoms caused by Host-Selective Toxins Molecules
secreted by the fungus
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Host-Selective Toxins
Produced by fungiSmall molecular weight moleculesProteins
Reproduce symptoms of diseasePathogen not required
Primary determinants of pathogenicity Toxic only to susceptible plants
Non-host plants not affected
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Symptoms of HSTs
Necrosis Cell death
Chlorosis Breakdown of chlorophyll
Necrotic Lesions
Chlorosis
Healthy Leaf
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HST of Ptr
ToxB Protein Causes chlorosis Multiple copies of the gene cause greater
chlorosis Inactive form toxb
Zone of Infiltration
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Hypothesis
A specific region of the structure of ToxB is responsible for the toxicity
of the protein.
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Project Goals
Purify ToxB from Ptr
MutagenesisImportant regions
of the gene for toxicity
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First Goal
Purification of ToxB
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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
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Purification Results
Protein Analysis Western Blot Silver Stain BCA Protein
Assay
Additional purification needed
P F1 F2 W1 W2 W3 M E1 E2 E3 E4
ToxB
75
50
25
20
10
15
37
kD
Silver Stain
Western Blot
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Second Goal
Mutagenesis of ToxB
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Sequence Comparison
Differences between the sequences Deletion in ToxB Proline in toxb
Restriction sites present in both Divides protein into three regions
toxb MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRCToxB MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC
C COOH
H
CH2
NH
H2
CH2C
I II III
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Chimeras
Chimeric ProteinsSwap Coding Regions
pCMR3 – BBbMAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC
pCMR4 – bbBMAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC
pCMR5 – BbbMAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC
pCMR6 – bBBMAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC
pCMR7 – BbBMAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC
pCMR8 – bBbMAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC
I II III
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pCMR2-ToxB3267 bp
ampicillin
ToxB
pUC ori
EcoRI (702)
NotI (1045)
BsaI (924)
BsaI (2414) BseYI (845)
BseYI (1764)
pCMR1-toxb3270 bp
ampicillin
toxb
pUC ori
EcoRI (702)
NotI (1048)
BsaI (924)
BsaI (2417) BseYI (845)
BseYI (1767)
ToxB/toxb Plasmids
Subcloned into pBSII One site each of
BseYI and BsaII
IIIII
III
III
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Expression of Chimeras
Subcloning into expression vector pPICZB
Transformation into Pichia pastorisExpression of chimerasSecretes protein
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Summary of Purification
Partial purification of ToxB
Next Steps of Purification
Finish purification Crystallization of ToxB 3-D Structure
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Summary of Mutagenesis
Generation of 5 of 6 chimeras
Two chimeras transformed into Pichia
BBb bbB Bbb bBB BbB bBb
BBb bbB Bbb bBB BbB bBb
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Next Steps of Mutagenesis
Finish generation of chimerasTransform into PichiaBioassayAdditional chimeras?
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Acknowledgements
HHMI URISC Dr. Lynda Ciuffetti Dr. P. Andrew Karplus Viola Manning Dr. Iovanna Pandelova Dr. Kevin Ahern