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Requirements for Ptr ToxA Entry into Sensitive Wheat Mesophyll Cells Sara M. Hamilton Viola A. Manning Dr. Lynda M. Ciuffetti Department of Botany and Plant Pathology Pyrenophora tritici-repentis Filamentous ascomycete Causes the disease tan spot of wheat Crop losses up to 50% worldwide Host-selective Toxins Pathogen Ptr ToxA • Ptr ToxB • Ptr ToxC • Ptr ToxD HST Ptr ToxA History First host-selective toxin (HST) isolated from P. tritici-repentis First proteinaceous HST identified Encoded by a single gene, ToxA Required for disease Ptr ToxA Causes necrosis on sensitive wheat cultivars Does not require pathogen to cause disease symptoms (HST slide) Reproduces disease symptoms in absence of pathogen Sensitive Insensitive Amino Acids Required for Disease Mutagenesis RGD cell attachment site within additional essential amino acids CKII phosphorylation site Crystal Structure Ganapathy Sarma and Dr. Andy Karplus Biochemistry & Biophysics Dept. OSU ToxA Internalization Green Fluorescent Protein Sensitive ToxA GFP-ToxA Ptr ToxA Insensitive Summary of Preliminary Data Amino acids required for disease: RGD cell attachment site and surrounding amino acids CKII site GFP-ToxA and confocal microscopy: Internalization into mesophyll cells Association with chloroplasts First protein shown to be internalized into plant cells from the apoplastic space What amino acids are involved in ToxA internalization? GFP-ToxA Mutant Proteins Mutagenize parent GFP-ToxA plasmid: Site-directed mutagenesis Subclone from previously mutagenized ToxA constructs PCR site-directed mutagenesis was more efficient GFP-ToxA Mutant Proteins Mutation to Alanine Method of Mutagenesis Motif Mutagenized Activity t66 site-directed PKC Active n76 site-directed Essential A.A. Not active v78 site-directed Essential A.A. Not active t79 site-directed CKII Not active r80 site-directed RGD Not active g81 site-directed RGD Not active d82 subcloned RGD Not active v83 site-directed Essential A.A. Not active GFP-ToxA Mutant Proteins Expressed in E.coli n76 Purified on a column SDS-PAGE 1 µg v78 t79 r80 g81 d82 v83 t66 GFP-ToxA mutant proteins = 55kDa Proteinase K Assay Infiltrate ToxA Incubate for 2hrs Infiltrate PK Incubate for 1hr Extract total protein Isolate ToxA Western Blot Proteinase K Assay PK TA TA TA TA TA TA TA TA TA TA Sensitive TA PK TA TA TA TA TA TA Insensitive ToxA Internalization into Cells of Sensitive Leaves ONLY Western blot with anti-ToxA antibody insensitive sensitive ToxA Proteinase K Assay GFP- PK TA TA GFPTA TA TA GFPTA GFPTA TA GFPTA GFPTA GFPTA TA GFPTA GFPTA TA TA GFP-ToxA Internalized PK GFPTA GFPTA GFPTA GFPTA GFPTA GFPTA GFPTA GFP-ToxA Not Internalized Proteins with Mutations in the RGD Loop are NOT Internalized ToxA GFP-ToxA A A A = Active protein I = Inactive protein n76 I t77 v78 I I t79 I r80 g81 d82 I I I v83 I t66 A Conclusions GFP-ToxA acts like ToxA Thus, it is a great tool Active ToxA is internalized ToxA internalization is required for disease Amino acids required for cell entry: RGD and essential aa’s CKII phosphorylation site Acknowledgements Dr. Lynda M. Ciuffetti HHMI Dr. Kevin Ahern Viola A. Manning Dr. Iovanna Pandelova Rachael Andrie Kristin Skinner Alex Babinin Dr. Andy Karplus Ganapathy Sarma Questions?
