Requirements for Ptr ToxA Entry into Sensitive Wheat Mesophyll Cells

1. 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

2. Pyrenophoratritici-repentis • Filamentous ascomycete • Causes the disease tan spot of wheat • Crop losses up to 50% worldwide

3. PtrToxB • PtrToxC • PtrToxD Host-selective Toxins Pathogen HST • PtrToxA

4. 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

5. Sensitive Insensitive 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

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

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

8. 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

9. What amino acids are involved in ToxA internalization?

10. 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

11. GFP-ToxA Mutant Proteins

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

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

14. PK TA TA TA TA TA TA TA PK Proteinase K Assay TA TA TA TA TA TA TA TA TA TA Sensitive TA TA TA TA TA TA TA Insensitive

15. Western blot with anti-ToxA antibody insensitive sensitive ToxA ToxA Internalization into Cells of Sensitive Leaves ONLY

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

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

18. 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

19. 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 Acknowledgements

20. Questions?