Kara Miles-Rockenfield In collaboration with the labs of Drs. Lynda Ciuffetti 1 and P. Andrew Karplus 2

1. 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 Ciuffetti1 and P. Andrew Karplus2 1Department of Botany and Plant Pathology 2Department of Biochemistry and Biophysics

2. 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!

3. Pyrenophora tritici-repentis • Fungus that causes tan-spot in wheat • Symptoms caused by Host-Selective Toxins • Molecules secreted by the fungus

4. 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

5. Symptoms of HSTs • Necrosis • Cell death • Chlorosis • Breakdown of chlorophyll Necrotic Lesions Healthy Leaf Chlorosis

6. Zone of Infiltration HST of Ptr • ToxB • Protein • Causes chlorosis • Multiple copies of the gene cause greater chlorosis • Inactive form toxb

7. Hypothesis A specific region of the structure of ToxB is responsible for the toxicity of the protein.

8. Project Goals • Purify ToxB from Ptr • Mutagenesis • Important regions of the gene for toxicity

9. First Goal Purification of ToxB

10. Plugs vs. Ground Filters 2-Step (NH4)2SO4 Cation Exchange Protein Precipitations Silver Stain, Western Blot, Protein Assay Purification of ToxB Protein Production in Crude Culture Filtrate Harvest Filtrate Chromatography Fraction Analysis

11. P F1 F2 W1 W2 W3 M E1 E2 E3 E4 kD 75 50 37 25 20 15 10 ToxB Purification Results • Protein Analysis • Western Blot • Silver Stain • BCA Protein Assay • Additional purification needed Western Blot Silver Stain

12. Mutagenesis of ToxB Second Goal

13. H2C H2C CH2 N C COOH H H Sequence Comparison I II III • Differences between the sequences • Deletion in ToxB • Proline in toxb • Restriction sites present in both • Divides protein into three regions toxbMAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC ToxBMAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC

14. pCMR3 – BBb MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC pCMR4 – bbB MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC pCMR5 – Bbb MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC pCMR6 – bBB MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC pCMR7 – BbB MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGNLIIRVGSDHSYLIRATVSCGLSL-TNQVFINGESVQSGGRC pCMR8 – bBb MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGELRIFVGSSHSYLIKATSSCGLSLNPSQSFINGESLASGGRC Chimeras • Chimeric Proteins • Swap Coding Regions I II III

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

16. Expression of Chimeras • Subcloning into expression vector • pPICZB • Transformation into Pichia pastoris • Expression of chimeras • Secretes protein

17. Summary of Purification • Partial purification of ToxB Next Steps of Purification • Finish purification • Crystallization of ToxB • 3-D Structure

18. 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

19. Next Steps of Mutagenesis • Finish generation of chimeras • Transform into Pichia • Bioassay • Additional chimeras?

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