Kara Miles-Rockenfield In collaboration with the labs of Drs. Lynda Ciuffetti 1 and P. Andrew Karplus 2 Purification, Crystallization, and Mutagenesis of ToxB, a Host- Selective Toxin in Pyrenophora tritici-repentis 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 …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 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  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  Mutagenesis  Important regions of the gene for toxicity

First Goal 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  Protein Analysis  Western Blot  Silver Stain  BCA Protein Assay  Additional purification needed P F1 F2 W1 W2 W3 M E1 E2 E3 E4 ToxB 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 MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC ToxB MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC CCOOH H CH 2 N H H2CH2C H2CH2C IIIIII

Chimeras  Chimeric Proteins  Swap Coding Regions pCMR3 – BBb MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC pCMR4 – bbB MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC pCMR5 – Bbb MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC pCMR6 – bBB MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC pCMR7 – BbB MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC pCMR8 – bBb MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC IIIIII

ToxB/toxb Plasmids  Subcloned into pBSII  One site each of BseYI and BsaI I II III I II III

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 Two chimeras transformed into Pichia BBbbbBBbbbBBBbBbBb BBbbbBBbbbBBBbBbBb

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