1. Analysis of the Site-of-Action and Evolution of the Host-Selective Toxin Ptr ToxB Wade Holman Dr. Lynda Ciuffetti’s Lab Department of Botany and Plant Pathology Oregon State University

2. Host-Selective Toxins (HST): Only produced by fungi Primary determinants of pathogenicity Reproduce symptoms of disease Pyrenophora tritici-repentis (Ptr) Disease: Tan Spot of Wheat

3. Host-Selective Toxins (HST)s of Ptr Protein Toxins –ToxA Necrosis –ToxB Chlorosis

4. ToxB characteristics 261 bp Open Reading Frame (ORF) 87 aa (8.9 kDa) S 23 aa- Signal sequence S Mature Ptr ToxB 64 aa (6.5 kDa) Multiple-copy gene ToxB preprotein

5. It’s still unknown Chlorophyll degradation Activity requires light ToxB sequence does not provide insights into the toxin’s mode or site-of-action. ToxB Mode-of-action?

6. Objectives To determine whether Ptr ToxB is internalized into the toxin sensitive cell To determine if Ptr ToxB homologs are present in different ascomycete species

7. Experimental approach Proteinase K Protection Assay (PK assay) Expression and Purification of ToxB and His ToxB Test activity of toxins

8. Purification of Pichia pastoris expressed ToxB and His-ToxB BCA assay/Adjust ToxB concentration to 15 µM KD QMA fractions containing contaminant- free ToxB collected during purification process. 10 15 20 37 250 50 Pichia pastoris culture concentration/dialysis of collected proteins QMA column

9. Activity of toxins

10. Proteinase K Protection assay TB PK Not Internalized TB PK Internalized TB PK=Proteinase K TB=ToxB Mesophyll Cells

11. Toxin-sensitive wheat leaf 2 h Tissue grinding and protein extraction Ni-NTA Protein Purification System Protein Gels For silver stain and western blot His-ToxB infiltration PK infiltration Symptom observation Chlorosis was monitored over 5 days Compared to the extracted protein results

12. Proteinase K Protection Assay using Pichia pastoris ToxB Assay shows a time dependent PK degradation effect on ToxB ToxB PK PK Time Points

13. Proteinase K Protection Assay using Pichia pastoris His-ToxB 24 h His-ToxB extraction Water Control His-ToxB Only His-ToxB+24h PK PK Only Symptom Observation Assay shows there is not PK degradation effect on His-ToxB PK: - + 10 kD 15 kD 20 kD His-ToxB

14. PK not working? High concentration of His-ToxB? PK Assay Troubleshooting

15. In vitro digestion of His-ToxB PMSF is a protease inhibitor Four tubes for in vitro digestion In vitro digestions went for 30 mins. at room temperature PMSF: His-ToxB: PK: + + -- ++ + + -+ +-

16. + PMSF No PK + PMSF PK - PMSF PK - PMSF No PK 20kD 15 kD 10 kD PK is active, although His-ToxB shows resistance to degradation. In vitro digestion of His-ToxB His-ToxB

17. Effect of PK concentrations on symptoms caused by His-ToxB PK causes chlorosis when infiltrated Exacerbates His-ToxB symptoms on leaves Difficult to determine if yellowing is due to toxin or PK His-ToxB + + +++ 150 ug/ml 200 ug/ml 300 ug/ml 400 ug/ml PK- ++ ++

18. Ideal concentration is between 150 and 200 ug/ml 300 and 400 ug/ml develop extensive chlorosis PK adjusted to 150 µg/ml in subsequent experiments Water His-ToxB PK 150 ug/ml+His-ToxB PK 200 ug/ml+His-ToxB PK 300 ug/ml+His-ToxB PK 400 ug/ml+His-ToxB

19. Screening for the presence of Ptr ToxB homologous sequences in different Ascomycete species Ptr ToxB homologs have been found in Pyrenophora bromi, a sister taxon to Ptr Pb Bf-1lg Pb TAM115 Pb Bf-1sm Pb SM20Alg Pb SM20Asm Pb SM101sm Pb SM106sm Pb SM106lg Pb TW123 Pb MPKlg Pb MPKsm Pb SM101lg Ptr toxb Ptr ToxB Global Identical Similar Normal Residue Key Amino acid alignments of Ptr ToxB, Ptr toxb, and ToxB homologs from different P. bromi isolates (Pb) (Andrie et.al., 2007)

20. Identification of ToxB homologs will be carried out by PCR. Screening of several ascomycete isolates Primers have been designed for the Internal Transcribed Spacer (ITS) sequence and the Ptr ToxB sequence within the ORF The ITS regions will serve as positive controls

21. PCR Results Fig. 1:PCR for ToxB gene with positive ToxB homolog in lane 6 Fig. 4: PCR for ITS regions Some isolates were too low in concentration for PCR amplification Two possible ToxB homologs were identified in this experiment 1 kB 500 bp 200 bp 1 kB 500 bp

22. = Pyrenophora tritici-repentis = Pleospora herbarum = Hysterium pulicare 29 isolates total were screened Of those, 2 possible homologs were identified One homolog was closely related to Ptr and one was distally related

23. Future Research To try PK protection assay with a more specific protease To screen the remaining isolates for ToxB To perform nested primer PCR on isolates I have already screened To locate possible ToxA homologs in ascomycetes

24. Acknowledgements Dr. Lynda Ciuffetti Dr. Melania Betts M.Sc. Viola Manning Dr. Iovanna Pandelova Dr. Tom Wolpert’s Lab Ernest and Pauline Jaworski Fund HHMI and Kevin Ahern OSU Department of Botany and Plant Pathology

25. Questions?