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.

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