Generation of Reactive Oxygen
Species in Wheat with
Treatment of Ptr ToxA, a HostSelective 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
 Introduction
 Hypothesis
 Strategy
 Summary
Pyrenophora tritici-repentis (Ptr)
 Fungal plant pathogen
 Causes the disease tan spot of wheat
 Crop losses of up to 50%
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
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
 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?
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
-,
.
Detection of ROS - H2O2
Substrate
Product
H2O2
H2DCFDA
DCF
Monitor
Fluorescence
Experimental Process
Infiltrate
Freeze
Process
Assay
Relative
Fluorescence Units
Fluorescence
Protein Concentration
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
Sensitive Plant
ROS in Insensitive Plants
 Does ROS accumulation occur in ToxA-infiltrated
insensitive plants?
Sensitive Plant
Relative
Fluorescence Units
Relative
Fluorescence Units
Insensitive Plant
Light-Dependency
Relative
Fluorescence Units
Relative
Fluorescence Units
 Sensitive and insensitive plants infiltrated
 One set in light for 24 hours
 One set in dark for 24 hours
ROS Scavengers Reduce ROS
Accumulation and Necrosis
Percent Control
 Scavenger = Ascorbic Acid
 Ascorbic Acid added 12 hours
post-toxin infiltration
 ROS measured 24 hours posttoxin infiltration
Necrosis visibly reduced!
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
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
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