Purification, Crystallization, and Mutagenesis of ToxB, a Host Selective Toxin in Pyrenophora tritici-repentis Kara Miles-Rockenfield In collaboration with the labs of Drs. Lynda Ciuffetti 1 and P. Andrew Karplus 2 1 Department of Botany and Plant Pathology 2 Department 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

Healthy Leaf Chlorosis

Symptoms of HSTs

 Necrosis  Cell death  Chlorosis  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 (NH 4 ) 2 SO 4

Chromatography

Cation Exchange

Fraction Analysis

Silver Stain, Western Blot, Protein Assay

Purification Results

Western Blot Silver Stain

25 20 15 10 kD P F1 F2 W1 W2 W3 M E1 E2 E3 E4 75 50 37

 Protein Analysis  Western Blot  Silver Stain  BCA Protein Assay  Additional purification needed

ToxB

Second Goal

Mutagenesis of

ToxB

Sequence Comparison

I II III

toxb ToxB MAPIF E TAMLLAVAILPAALVSANC T ANILNINE V VIATGCVPAGG NLI I R VGS D HSYLI R ATVSCGLSL NPS QSFINGES LA SGGRC MAPIF K TTMLLAVAILPAALVSANC V ANILNINE A VIATGCVPAGG ELR I F VGS S HSYLI K ATSSCGLSL -TN QVFINGES VQ SGGRC

  Differences between the sequences  Deletion in

ToxB

 Proline in

toxb

Restriction sites present in both  Divides protein into three regions H 2 C N H 2 C C CH 2 COOH H H

Chimeras

 Chimeric Proteins  Swap Coding Regions

pCMR3 – BBb

I II

MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCG

III

LSLNPSQSFINGESLASGGRC pCMR4 – bbB MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCG LSL-TNQVFINGESVQSGGRC pCMR5 – Bbb MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGC VPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC pCMR6 – bBB MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGC VPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC pCMR7 – BbB MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGC VPAGGNLIIRVGSDHSYLIRATVSCG LSL-TNQVFINGESVQSGGRC pCMR8 – bBb MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGC VPAGGELRIFVGSSHSYLIKATSSCG LSLNPSQSFINGESLASGGRC

ToxB

/

toxb

Plasmids

ampicillin

Bsa I (2417)

pUC ori

Bse YI (1767) pCMR1-toxb 3270 bp II III I Eco RI (702) Bse YI (845)

toxb

Bsa I (924) Not I (1048)

ampicillin

 Subcloned into pBSII  One site each of BseYI and BsaI Bsa I (2414) pCMR2-ToxB 3267 bp II I III Eco RI (702) 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

 Generation of 5 of 6 chimeras

BBb bbB Bbb bBB BbB bBb

 Two chimeras transformed into

Pichia

BBb 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