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 Filamentous ascomycete Causes the disease tan spot of wheat Causes the disease tan spot of wheat Crop losses up to 50% worldwide Crop losses up to 50% worldwide

Host-selective Toxins Ptr ToxC Ptr ToxD Ptr ToxB Pathogen HST Ptr ToxA Ptr ToxA

Ptr ToxA History First host-selective toxin (HST) isolated from P. tritici-repentis First host-selective toxin (HST) isolated from P. tritici-repentis First proteinaceous HST identified First proteinaceous HST identified Encoded by a single gene, ToxA Encoded by a single gene, ToxA Required for disease Required for disease

Ptr ToxA Causes necrosis on sensitive wheat cultivars Causes necrosis on sensitive wheat cultivars Does not require pathogen to cause disease symptoms (HST slide) Does not require pathogen to cause disease symptoms (HST slide) Reproduces disease symptoms in absence of pathogen Reproduces disease symptoms in absence of pathogen Sensitive Insensitive

Mutagenesis Mutagenesis RGD cell attachment site within additional essential amino acids RGD cell attachment site within additional essential amino acids CKII phosphorylation site CKII phosphorylation site Crystal Structure Crystal Structure Ganapathy Sarma and Dr. Andy Karplus Biochemistry & Biophysics Dept. OSU Amino Acids Required for Disease

ToxA GFP-ToxA ToxA Internalization Green Fluorescent Protein Ptr ToxA Insensitive Sensitive

Summary of Preliminary Data Amino acids required for disease: Amino acids required for disease: RGD cell attachment site and RGD cell attachment site and surrounding amino acids CKII site CKII site GFP-ToxA and confocal microscopy: GFP-ToxA and confocal microscopy: Internalization into mesophyll cells Internalization into mesophyll cells Association with chloroplasts Association with chloroplasts First protein shown to be internalized into plant cells from the apoplastic space 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: Mutagenize parent GFP-ToxA plasmid: Site-directed mutagenesis Site-directed mutagenesis Subclone from previously mutagenized ToxA constructs Subclone from previously mutagenized ToxA constructs PCR site-directed mutagenesis was more efficient PCR site-directed mutagenesis was more efficient

Mutation to Alanine Method of Mutagenesis Motif Mutagenized Activity t66site-directed PKC PKCActive n76site-directed Essential A.A. Not active v78site-directed Essential A.A. Not active t79site-directedCKII r80site-directedRGD g81site-directedRGD d82subclonedRGD v83site-directed Essential A.A. Not active GFP-ToxA Mutant Proteins

Expressed in E.coli Expressed in E.coli Purified on a column Purified on a column SDS-PAGE SDS-PAGE 1 µg 1 µg n76 v78 t79 r80 g81 d82 v83 t66 n76 v78 t79 r80 g81 d82 v83 t66 GFP-ToxA mutant proteins = 55kDa

Proteinase K Assay Infiltrate ToxA Infiltrate ToxA Incubate for 2hrs Incubate for 2hrs Infiltrate PK Infiltrate PK Incubate for 1hr Incubate for 1hr Extract total protein Extract total protein Isolate ToxA Isolate ToxA Western Blot Western Blot

Proteinase K Assay TA PK Insensitive TA PK Sensitive TA

ToxA Internalization into Cells of Sensitive Leaves ONLY insensitivesensitive ToxA Western blot with anti-ToxA antibody

Proteinase K Assay PK TA PK GFP-ToxA Internalized GFP- TA GFP-ToxA Not Internalized GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA GFP- TA

Proteins with Mutations in the RGD Loop are NOT Internalized ToxA A GFP-ToxA A t66 A n76 t77 v78 t79 I r80 g81 d82 v83 IIIIIII A = Active protein I = I nactive protein

Conclusions GFP-ToxA acts like ToxA GFP-ToxA acts like ToxA Thus, it is a great tool Thus, it is a great tool Active ToxA is internalized Active ToxA is internalized ToxA internalization is required for disease ToxA internalization is required for disease Amino acids required for cell entry: Amino acids required for cell entry: RGD and essential aa’s RGD and essential aa’s CKII phosphorylation site CKII phosphorylation site

Acknowledgements Dr. Lynda M. Ciuffetti Dr. Lynda M. Ciuffetti HHMI HHMI Dr. Kevin Ahern Dr. Kevin Ahern Viola A. Manning Viola A. Manning Dr. Iovanna Pandelova Dr. Iovanna Pandelova Rachael Andrie Rachael Andrie Kristin Skinner Kristin Skinner Alex Babinin Alex Babinin Dr. Andy Karplus Dr. Andy Karplus Ganapathy Sarma Ganapathy Sarma

Questions?