Generation of Reactive Oxygen Species in Wheat with Treatment of Ptr ToxA, a Host-Selective Toxin

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 • Introduction • Hypothesis • Strategy • Summary

4. Pyrenophoratritici-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. Sensitive Insensitive Ptr ToxA • Causes necrosis on sensitive wheat cultivars • Does not require pathogen to cause disease symptoms • Reproduces disease symptoms in absence of pathogen

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 H2O2, O2-, OH.

9. Detection of ROS - H2O2 Substrate Product H2O2 DCF H2DCFDA Monitor Fluorescence

10. Experimental Process Freeze Process Infiltrate Assay Relative Fluorescence Units Fluorescence Protein Concentration

11. Sensitive Plant Relative Fluorescence Units 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

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

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

14. Percent Control 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 • 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