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 Laboratory
Department 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
ToxB necrosis (cell death) chlorosis (breakdown of chlorophyll)
Ptr ToxA 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)
G

Reactive Oxygen Species
Hydrogen Peroxide – H
2
O
2
Superoxide – O
2
-
• Highly reactive due to unpaired valence electron
• Bi-product of several metabolic pathways
• Often produced in stressful environment
• Targeted by anti-oxidants
OH attacking DNA

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

• 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

Infiltration
ROS Detection Procedure
Freeze using N
2
Centrifugation
Measurement of fluorescence
• After N
2 treatment, the leaf tissue is ground and suspended in Tris-HCl
• After centrifugation, supernatant is removed and H
2
DCFDA is added
•H
2
DCFDA fluoresces in the presence of ROS
ROS
H
2
DCFDA DCF

ROS Quantification
3
2,5
2
1,5
1
0,5
0
0,17
ROS Production vs. Treatments
2,7
0,11
0,98
No Treatment Water ToxA ToxB
•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 O
2
-
NBT

ROS Visualization using NBT
ToxB H
2
O
Sensitive
Insensitive
• 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

1. To determine whether the accumulation of reactive oxygen species plays a role in the mode-of-action of ToxB
– ROS include H
2
O
2,
O
2
-
,
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

Extraction and quantification of phenolic compounds
Infiltration Freeze using N
2
Centrifugation
Absorbtion at
725 nm
• After N
2
CH
3
OH treatment, the leaf tissue is ground and suspended in 50%
• 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
Quantification of free phenolics in toxin sensitive (Katepwa) and insensitive (Auburn) wheat cultivars
2
1,8
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
S
Water
I S
ToxA
I
24 hours
S
ToxB
I S
Water
I S
ToxA
I
48 hours
S
ToxB
I
• 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

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 College
HHMI committee
Dr. Lynda Ciuffetti
Dr. Melania Betts
Dr. Iovanna Pandelova
Viola Manning