1. Department of Botany and Plant Pathology Oregon State University
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

2. 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)

3. 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

4. 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
Ptr ToxB
Sensitive
Insensitive

5. Mode-of-action of Ptr ToxAG D
Internalizes in ToxA sensitive mesophyll cells
Localizes to the chloroplast
Light dependent
Disruption of photosynthesis
Accumulation of high levels of reactive oxygen species (ROS)

6. 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

7. 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

8. 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

9. ROS Detection Procedure
Measurement of fluorescence
Infiltration
Freeze using N2
Centrifugation
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
ROS
H2DCFDA
DCF

10. 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

11. 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

12. ROS Visualization using NBT
ToxB
H2O
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

13. 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

14. Accumulation of phenolics is a typical response to plant pathogens
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

15. Extraction and quantification of phenolic compounds
Absorbtion at 725 nm
Infiltration
Freeze using N2
Centrifugation
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

16. ToxA produced phenolics at a higher rate than ToxB
Quantification of free phenolic compounds in ToxA and ToxB treated plants S I S I S I S I S I S I
Water
ToxA
ToxB
Water
ToxA
ToxB
24 hours
48 hours
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

17. 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

18. Acknowledgements Dr. Kevin Ahern University Honors College
HHMI committee
Dr. Lynda Ciuffetti
Dr. Melania Betts
Dr. Iovanna Pandelova
Viola Manning