1. Generation of Reactive Oxygen Species in Wheat with Treatment of Ptr ToxA, a Host- Selective Toxin Joshua E. Steeves Viola A. Manning Dr. Lynda Ciuffetti Department of Botany and Plant Pathology

2. Reactive Oxygen Species  Important role in humans and plants

3. Generation of ROS in Wheat Generation of ROS in Wheat  Introduction  Hypothesis  Strategy  Summary

4. Pyrenophora tritici-repentis (Ptr)  Fungal plant pathogen  Causes the disease tan spot of wheat  Crop losses of up to 50%

5. Ptr ToxA History  First host-selective toxin (HST) isolated from P. tritici-repentis   Produced only by Fungi   Reproduce Symptoms of Disease   Primary Determinants of Pathogenicity   Toxic only to Susceptible Plants  First proteinaceous HST described  Required for disease

6. Ptr ToxA  Causes necrosis on sensitive wheat cultivars  Does not require pathogen to cause disease symptoms  Reproduces disease symptoms in absence of pathogen Sensitive Insensitive

7.  Ptr ToxA localizes to chloroplasts and affects photosynthesis (Manning et. al 2004)  Disruption of photosynthesis can produce high levels of Reactive Oxygen Species (ROS)  High levels of ROS lead to necrosis  Does Ptr ToxA cause necrosis via the accumulation of ROS?

8. ROS in Plants  Byproduct of normal metabolism  Cellular levels controlled by enzymes and antioxidants  Biotic and abiotic stresses increase ROS production  ROS can act as signaling molecules  ROS include H 2 O 2, O 2 -, OH.

9. Detection of ROS - H 2 O 2 H 2 DCFDA Substrate Product DCF H2O2H2O2 Monitor Fluorescence

10. Experimental Process Infiltrate FreezeProcess Assay FluorescenceProtein Concentration Relative Fluorescence Units

11. Time Course  What is the time course of ROS accumulation in ToxA-treated sensitive plants?  1, 3, 6, 9, 12, 18, and 24 hour post-infiltration Relative Fluorescence Units Sensitive Plant

12. ROS in Insensitive Plants  Does ROS accumulation occur in ToxA-infiltrated insensitive plants? insensitive plants? Relative Fluorescence Units Insensitive Plant Relative Fluorescence Units Sensitive Plant

13. Light-Dependency  Sensitive and insensitive plants infiltrated  One set in light for 24 hours  One set in dark for 24 hours Relative Fluorescence Units Relative Fluorescence Units

14. ROS Scavengers Reduce ROS Accumulation and Necrosis  Scavenger = Ascorbic Acid  Ascorbic Acid added 12 hours post-toxin infiltration  ROS measured 24 hours post- toxin infiltration Percent Control  Necrosis visibly reduced!

15. Conclusions  ToxA treatment leads to accumulation of ROS in sensitive wheat  Accumulation of ROS is correlated with necrosis  ROS accumulation requires light as does necrosis  Addition of ROS scavengers reduces ROS levels and necrosis

16. Implications and Future Directions  These data imply that ROS is the cause of ToxA-induced necrosis  Future Experiments  Where are ROS being generated?  What species of ROS are generated?  What comes first: the chicken or the egg?  decrease in photosynthesis or increase in ROS

17. Acknowledgments  The Howard Hughes Medical Institute  Dr. Kevin Ahern  Viola Manning  Dr. Lynda Ciuffetti  The Ciuffetti Lab  Iovanna Pandelova  Kristin Skinner  Sara Hamilton  Josh Cuperus