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

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. Reactive Oxygen Species. Important role in humans and plants. Generation of ROS in Wheat.

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

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  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

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