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

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)

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 fungiSmall molecular weight moleculesProteins

Reproduce symptoms of diseasePathogen not required

Primary determinants of pathogenicity Toxic only to susceptible plants

Non-host plants not affected

Symptoms of HSTs

Necrosis Cell death

Chlorosis Breakdown of chlorophyll

Necrotic Lesions

Chlorosis

Healthy Leaf

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

MutagenesisImportant 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

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

Second Goal

Mutagenesis of ToxB

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

Chimeras

Chimeric ProteinsSwap Coding Regions

pCMR3 – BBbMAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC

pCMR4 – bbBMAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC

pCMR5 – BbbMAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC

pCMR6 – bBBMAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC

pCMR7 – BbBMAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC

pCMR8 – bBbMAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC

I II III

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

Expression of Chimeras

Subcloning into expression vector pPICZB

Transformation into Pichia pastorisExpression of chimerasSecretes protein

Summary of Purification

Partial purification of ToxB

Next Steps of Purification

Finish purification Crystallization of ToxB 3-D Structure

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

Next Steps of Mutagenesis

Finish generation of chimerasTransform into PichiaBioassayAdditional chimeras?

Acknowledgements

HHMI URISC Dr. Lynda Ciuffetti Dr. P. Andrew Karplus Viola Manning Dr. Iovanna Pandelova Dr. Kevin Ahern