generation of reactive oxygen species in wheat with treatment of ptr toxa, a host- selective toxin...
TRANSCRIPT
Generation of Reactive Oxygen Generation of Reactive Oxygen Species in Wheat with Species in Wheat with
Treatment of Ptr ToxA, a Host-Treatment of Ptr ToxA, a Host-Selective ToxinSelective Toxin
Joshua E. SteevesJoshua E. SteevesViola A. ManningViola A. ManningDr. Lynda CiuffettiDr. Lynda Ciuffetti
Department of Botany and Plant PathologyDepartment of Botany and Plant Pathology
Reactive Oxygen SpeciesReactive Oxygen Species
Important role in humans and plantsImportant role in humans and plants
Generation of ROS in WheatGeneration of ROS in Wheat
IntroductionIntroduction
HypothesisHypothesis
StrategyStrategy
SummarySummary
PyrenophoraPyrenophora triticitritici--repentis (Ptr)repentis (Ptr)
Fungal plant pathogen Fungal plant pathogen
Causes the disease tan spot of wheatCauses the disease tan spot of wheat
Crop losses of up to 50%Crop losses of up to 50%
Ptr ToxA HistoryPtr ToxA HistoryFirst host-selective toxin (HST) isolated First host-selective toxin (HST) isolated
from from P. tritici-repentisP. tritici-repentis Produced only by Fungi Reproduce Symptoms of Disease Primary Determinants of Pathogenicity Toxic only to Susceptible Plants
First proteinaceous HST describedFirst proteinaceous HST described
Required for diseaseRequired for disease
Ptr ToxAPtr ToxA
Causes necrosis on sensitive wheat cultivarsCauses necrosis on sensitive wheat cultivars
Does not require pathogen to cause disease Does not require pathogen to cause disease symptomssymptoms
Reproduces disease symptoms in absence of Reproduces disease symptoms in absence of pathogenpathogen
Sensitive
Insensitive
Ptr ToxA localizes to chloroplasts and Ptr ToxA localizes to chloroplasts and affects photosynthesis affects photosynthesis (Manning et. al 2004)(Manning et. al 2004)
Disruption of photosynthesis can Disruption of photosynthesis can produce high levels of Reactive Oxygen produce high levels of Reactive Oxygen Species (ROS)Species (ROS)
High levels of ROS lead to necrosisHigh levels of ROS lead to necrosis
Does Ptr ToxA cause necrosis via Does Ptr ToxA cause necrosis via the accumulation of ROS?the accumulation of ROS?
ROS in PlantsROS in Plants Byproduct of normal metabolismByproduct of normal metabolism
Cellular levels controlled by enzymes and Cellular levels controlled by enzymes and antioxidantsantioxidants
Biotic and abiotic stresses increase ROS Biotic and abiotic stresses increase ROS productionproduction
ROS can act as signaling molecules ROS can act as signaling molecules
ROS include HROS include H22OO22, O, O22--, OH, OH..
Detection of ROS - HDetection of ROS - H22OO22
H2DCFDA
Substrate Product
DCF
H2O2
Monitor Fluorescence
Experimental ProcessExperimental Process
Infiltrate Freeze Process
Assay
Fluorescence Protein Concentration
RelativeFluorescence Units
Time CourseTime Course What is the time course of ROS accumulation in What is the time course of ROS accumulation in
ToxA-treated sensitive plants?ToxA-treated sensitive plants? 1, 3, 6, 9, 12, 18, and 24 hour post-infiltration1, 3, 6, 9, 12, 18, and 24 hour post-infiltration
Sensitive
0 5 10 15 20 25 30-20
0
20
40
60
80
100
120
WaterToxA
Time Post-infiltration (Hour)
Rel
ativ
e F
luo
resc
ence
Un
its
Sensitive Plant
ROS in Insensitive PlantsROS in Insensitive Plants Does ROS accumulation occur in ToxA-infiltrated Does ROS accumulation occur in ToxA-infiltrated insensitive plants?insensitive plants?
Insensitive
0 5 10 15 20 25 30-20
0
20
40
60
80
100
120
WaterToxA
Time Post-infiltration (Hour)
Rel
ati
ve
Flu
ore
scen
ce
Un
its
Insensitive Plant Sensitive
0 5 10 15 20 25 30-20
0
20
40
60
80
100
120
WaterToxA
Time Post-infiltration (Hour)
Rel
ati
ve
Flu
ore
scen
ce
Un
its
Sensitive Plant
Light-DependencyLight-Dependency Sensitive and insensitive plants infiltratedSensitive and insensitive plants infiltrated
One set in light for 24 hoursOne set in light for 24 hours One set in dark for 24 hoursOne set in dark for 24 hours
Insensitive Plant
ToxA Water0
10
20
30
40
50
60
70
LightDark
Treatment
Rel
ati
ve
Flu
ore
scen
ce
Un
its
Sensitive Plant
ToxA Water0
10
20
30
40
50
60
70
LightDark
Treatment
Rel
ati
ve
Flu
ore
scen
ce
Un
its
ROS Scavengers Reduce ROS ROS Scavengers Reduce ROS Accumulation and NecrosisAccumulation and Necrosis
Scavenger = Ascorbic Acid
Ascorbic Acid added 12 hours
post-toxin infiltration
ROS measured 24 hours post-
toxin infiltrationToxA ToxA + Scavenger0
25
50
75
100
Treatment
Pe
rce
nt
Co
ntr
ol
Necrosis visibly reduced!
ConclusionsConclusions
ToxA treatment leads to accumulation ToxA treatment leads to accumulation of ROS in sensitive wheatof ROS in sensitive wheat
Accumulation of ROS is correlated with Accumulation of ROS is correlated with necrosisnecrosis
ROS accumulation requires light as ROS accumulation requires light as does necrosisdoes necrosis
Addition of ROS scavengers reduces Addition of ROS scavengers reduces ROS levels and necrosisROS levels and necrosis
Implications and Future DirectionsImplications and Future Directions
These data imply that ROS is the cause of These data imply that ROS is the cause of ToxA-induced necrosisToxA-induced necrosis
Future ExperimentsFuture Experiments
Where are ROS being generated?Where are ROS being generated?
What species of ROS are generated?What species of ROS are generated?
What comes first: the chicken or the egg?What comes first: the chicken or the egg?
decrease in photosynthesis or increase in ROSdecrease in photosynthesis or increase in ROS
AcknowledgmentsAcknowledgments
The Howard Hughes Medical InstituteThe Howard Hughes Medical Institute Dr. Kevin AhernDr. Kevin Ahern Viola ManningViola Manning Dr. Lynda CiuffettiDr. Lynda Ciuffetti The Ciuffetti LabThe Ciuffetti Lab
Iovanna PandelovaIovanna Pandelova Kristin SkinnerKristin Skinner Sara Hamilton Sara Hamilton Josh CuperusJosh Cuperus