Aakash Mankaney Ciuffetti Laboratory Department of Botany and Plant Pathology

Characterization of ROS production and cell wall modifications induced by the host-selective toxins of

Pyrenophora tritici-repentis Ptr ToxA and Ptr ToxB

Aakash Mankaney

Ciuffetti LaboratoryDepartment of Botany and Plant Pathology

Oregon State University

Pyrenophera-tritici repentis (Ptr)

• Plant fungal pathogen

• Causal agent of tan spot disease of wheat

• Responsible for destruction of 2% to 40% of wheat crop

• Produces host-selective toxins (HSTs)

Host-selective toxins

• HSTs are defined as a group of metabolites produced only by fungal pathogens

• Function as essential determinants of pathogenicity • In toxin sensitive plants, HSTs produce disease symptoms

in absence of pathogen that produces them

• Understanding the mode-of-action of HSTs will contribute towards designing strategies to prevent tan spot disease of wheat

Ptr ToxA (ToxA) and Ptr ToxB (ToxB)

• ToxA and ToxB are two proteinaceous HSTs isolated from P. tritici-repentis

• ToxA and ToxB can be heterologously expressed in Escherichia coli and Pichia pastoris, respectively

• ToxA necrosis (cell death)ToxB chlorosis (breakdown of chlorophyll)

Ptr ToxA

Sensitive

Insensitive

Ptr ToxB

Mode-of-action of Ptr ToxA

• Internalizes in ToxA sensitive mesophyll cells

• Localizes to the chloroplast • Light dependent

• Disruption of photosynthesis

• Accumulation of high levels of reactive oxygen species (ROS)

R G D

Reactive Oxygen Species

Hydrogen Peroxide – H2O2 Superoxide – O2- OH- attacking DNA

• Highly reactive due to unpaired valence electron

• Bi-product of several metabolic pathways

• Often produced in stressful environment

• Targeted by anti-oxidants

Does ToxB treatment induce the production of ROS in the sensitive cultivar?

• Requires light for symptom development

• ToxB is involved in photooxidation of chlorophyll, affecting its function

• We hypothesize that Ptr ToxB will increase the cellular levels of ROS

Objectives

• Determine if ToxB induces production of ROS in a sensitive wheat cultivar

• Compare the effect of ToxA and ToxB on basic plant defense responses on sensitive and insensitive wheat cultivars– Production of phenolic compounds

ROS Detection Procedure

Infiltration Centrifugation Freeze using N2

Measurement of fluorescence

• After N2 treatment, the leaf tissue is ground and suspended in Tris-HCl

• After centrifugation, supernatant is removed and H2DCFDA is added

• H2DCFDA fluoresces in the presence of ROS

H2DCFDA DCF

ROS

No Treatment Water ToxA ToxB0

0.5

1

1.5

2

2.5

3

0.17 0.11

2.7

0.98

ROS Production vs. Treatments

Rela

tive

Fluo

resc

ence

Uni

ts

ROS Quantification

• Sensitive wheat leaves infiltrated by water and no treatment produce roughly the same level of ROS

• Sensitive wheat leaves incubated with ToxA produce more ROS after 48 hours than sensitive wheat leaves incubated with ToxB

ROS Visualization using Nitro blue tetrazolium (NBT)

Infiltration NBT Staining Chlorophyll Extraction Visualization

• NBT forms a blue, formazen deposit when oxidized

• The blue deposit is viewed under a microscope

• NBT is oxidized by O2-

NBT

ToxB H2O

Sensitive

Insensitive

ROS Visualization using NBT

• Only the ToxB treated leaves contain formazan deposits

• ToxB treated sensitive leaves contain more formazan deposits than ToxB treated insensitive leaves

• Leaves treated with ToxB produce ROS-Sensitive leaves produce greater amounts of ROS

Objectives

1. To determine whether the accumulation of reactive oxygen species plays a role in the mode-of-action of ToxB– ROS include H2O2, O2

-, OH

2. To compare the effect of ToxA and ToxB on basic plant defense responses on sensitive and insensitive wheat cultivars– Production of phenolic compounds

Phenolic Compounds

• Phenolic compounds are secondary metabolites consisting of a hydroxyl group bonded to an aromatic hydrocarbon

• Accumulation of phenolics is a typical response to plant pathogens

• Phenolics are precursors to structural barriers

• Activate plant defense genes

• Include tyrosine, capsaicin, flavonoids and tannins

– Flavonoids, tannins, hydroxycinnamate

Extraction and quantification of phenolic compounds

Infiltration Centrifugation Freeze using N2

Absorbtion at 725 nm

• After N2 treatment, the leaf tissue is ground and suspended in 50% CH3OH

• After centrifugation, supernatant is removed and mixed with the Folin-Ciocalteau reagent

• After 20 minutes incubation, the absorption is measured at 725 nm, and total phenolics are quantified

Quantification of free phenolic compounds in ToxA and ToxB treated plants

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2Quantification of free phenolics in toxin sensitive (Katepwa) and insensitive (Auburn) wheat cultivars

Tota

l phe

nolic

s (u

g p-

coum

aric

aci

d pe

r m

g of

fres

h w

eigh

t)

S I S I S I IIIS S SToxB ToxBWater WaterToxA ToxA

• Only leaves treated with ToxA produced phenolic compounds 24 hours after treatment

• Leaves incubated with ToxB do not produce a significant level of phenolics at 24 hours, however production is greater at 48 hours

• ToxA produced phenolics at a higher rate than ToxB

24 hours 48 hours

Conclusions

• Leaves treated with ToxB produce ROS-Sensitive leaves produce greater amounts of ROS

• Both ToxA and ToxB induce production of phenolic compounds in sensitive wheat cultivars

• Sensitive leaves incubated with ToxA produce phenolic compounds at a faster rate than ToxB

• Phenolic production is greater in sensitive wheat cultivars at 48 hours of incubation verses 24 hours incubation

Dr. Kevin Ahern University Honors CollegeHHMI committee Dr. Lynda CiuffettiDr. Melania BettsDr. Iovanna PandelovaViola Manning

Acknowledgements