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Does Plant Cell Death Induced by Ptr ToxA Require Toxin Entry?

Does Plant Cell Death Induced by Ptr ToxA Require Toxin Entry?. Sara M. Hamilton Viola A. Manning Dr. Lynda M. Ciuffetti Department of Botany and Plant Pathology. Pyrenophora tritici - repentis. Filamentous fungus-ascomycete

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Does Plant Cell Death Induced by Ptr ToxA Require Toxin Entry?

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

  2. Pyrenophoratritici-repentis • Filamentous fungus-ascomycete • Plant pathogen causing the disease tanspot of sensitive wheat species • Crop losses estimated up to 50% in susceptible varieties worldwide

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

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

  5. Ptr ToxA • Causes necrosis on sensitive wheat cultivars • 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?

  8. Conserved ToxA Motifs • “RGD” cell attachment site • 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

  9. ToxA Protein Sequence QGSCMSITINPSRPSVNNIGQVDIDSVILG RPGAIGSWELNNFITIGLNRVNADTVRVNI RNTGRTNRLIITQWDNTVTRGDVYELFGDY ALIQGRGSFCLNIRSDTGRENWRMQLEN • 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)?

  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. Sensitive Insensitive ToxA GFP-ToxA GFP-ToxA: Localization to Chloroplasts

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

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

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

  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.

  17. Mutations of GFP-ToxA * Essential amino acids surround the RGD motif

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

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

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

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

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

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