1. Does Plant Cell Death Induced by Ptr ToxA Require Toxin Entry? Sara M. Hamilton Sara M. Hamilton Viola A. Manning Dr. Lynda M. Ciuffetti Department of Botany and Plant Pathology

2. Pyrenophora tritici-repentis Filamentous fungus-ascomycete Filamentous fungus-ascomycete Plant pathogen causing the disease tan spot of sensitive wheat species Plant pathogen causing the disease tan spot of sensitive wheat species Crop losses estimated up to 50% in susceptible varieties worldwide Crop losses estimated up to 50% in susceptible varieties worldwide

3. Races of Pyrenophora tritici-repentis N = causes necrosis N = causes necrosis C = causes chlorosis C = causes chlorosis R = resistant to pathogen R = resistant to pathogen Race 1 Race 2 Race 3 Race 4 Race 5 Salamouni Glenlea N (ToxA) R R R R R R R R Katepwa N (ToxA) R R C (ToxB) 6B365 C (ToxC) R R R 6B662 R R R R C (ToxB)

4. Ptr ToxA First host selective toxin (HST) isolated from P. tritici-repentis First host selective toxin (HST) isolated from P. tritici-repentis First proteinaceous HST First proteinaceous HST Encoded by a single gene, the ToxA gene Encoded by a single gene, the ToxA gene

5. Ptr ToxA Causes necrosis on sensitive wheat cultivars Causes necrosis on sensitive wheat cultivars Does not require pathogen to cause disease symptoms Does not require pathogen to cause disease symptoms Sensitive Insensitive

6. We want to know: 1. What part of the ToxA protein is necessary for disease symptoms? 2. Where does the protein exert activity (i.e. where is the site-of-action)?

7. Question #1 What part of the ToxA protein is necessary for disease? What part of the ToxA protein is necessary for disease?

8. Conserved ToxA Motifs “RGD” cell attachment site “RGD” cell attachment site RGD sites mediate interaction of cell matrix proteins with a family of membrane-bound receptors called integrins. RGD sites mediate interaction of cell matrix proteins with a family of membrane-bound receptors called integrins. Casein kinase II (CKII) and Protein kinase C (PKC) phosphorylation sites Casein kinase II (CKII) and Protein kinase C (PKC) phosphorylation sites

9. QGSCMSITINPSRPSVNNIGQVDIDSVILG RPGAIGSWELNNFITIGLNRVNADTVRVNI RNTGRTNRLIITQWDNTVTRGDVYELFGDY ALIQGRGSFCLNIRSDTGRENWRMQLEN ToxA Protein Sequence  Both the RGD and casein kinase II phosphorylation motifs are required for ToxA activity

10. Question #2 Where does the protein exert activity (i.e. where is the site-of-action)? Where does the protein exert activity (i.e. where is the site-of-action)?

11. ToxA Localization  ToxA is imported into mesophyll cells of sensitive wheat genotypes and localizes to the chloroplasts of these cells.  ToxA localization can be visualized in vivo by treatment of wheat with a green flourescent protein (GFP) fused to ToxA (GFP-ToxA).

12. GFP-ToxA: Localization to Chloroplasts SensitiveInsensitive ToxA GFP-ToxA

13. Hypothesis ToxA entry into mesophyll cells is required to cause cell death. ToxA entry into mesophyll cells is required to cause cell death.

14. Current Study Produce GFP-ToxA proteins harboring mutations and determine their localization in planta Produce GFP-ToxA proteins harboring mutations and determine their localization in planta Mutations include amino acids in the RGD cell attachment site and phosphorylation motifs. Mutations include amino acids in the RGD cell attachment site and phosphorylation motifs.

15. GFP-ToxA: Construction of Fusion Protein Vector Green Fluorescent Protein Ptr ToxA

16. GFP-ToxA Mutants  Mutagenize parent GFP-ToxA plasmid:  Site-directed mutagenesis  Subcloning from previously mutagenized ToxA constructs PCR site-directed mutagenesis proved to be more efficient than subcloning. PCR site-directed mutagenesis proved to be more efficient than subcloning.

17. Mutation to Alanine Method of MutagenesisMotif Mutagenized t63subclonedPKC t66site-directedPKC n76site-directed*Essential A.A. t77subcloned*Essential A.A. v78site-directed*Essential A.A. t79site-directedCKII r80site-directedRGD g81site-directedRGD d82subclonedRGD v83site-directed*Essential A.A. e85site-directed*Essential A.A. Mutations of GFP-ToxA * Essential amino acids surround the RGD motif

18. Expression of GFP-ToxA Transformation of E. coli with vector Transformation of E. coli with vector Expression of GFP-ToxA in E. coli Expression of GFP-ToxA in E. coli Purification of GFP-ToxA Purification of GFP-ToxA

19. Protein Purification kDa 24 72 33 40 55 pCVM77 fusion protein gel

20. To Be Completed: Infiltration of GFP-ToxA mutant proteins into sensitive/insensitive wheat leaves: Infiltration of GFP-ToxA mutant proteins into sensitive/insensitive wheat leaves: Assay activity Assay activity Determine localization via fluorescent microscopy Determine localization via fluorescent microscopy

21. Dissecting the ToxA Pathway This information will allow us to determine if the mutant proteins synthesized will: This information will allow us to determine if the mutant proteins synthesized will: Cross the cell wall Cross the cell wall Cross the plasma membrane Cross the plasma membrane Localize to an organelle (ex. chloroplast) Localize to an organelle (ex. chloroplast)

22. Acknowledgements Howard Hughes Medical Institution Howard Hughes Medical Institution Ernest and Pauline Jaworski Ernest and Pauline Jaworski USDA USDA Dr. Kevin Ahern Dr. Kevin Ahern Dr. Lynda M. Ciuffetti Dr. Lynda M. Ciuffetti Viola A. Manning Viola A. Manning  Dr. Pat Martinez  Dr. Iovanna Pandelova  Kristin Skinner  Rachael Andrie  Rebecca Tippner-Hedges  Alex Babinin