Aakash Mankaney Ciuffetti Laboratory Department of Botany and Plant Pathology

1. Characterization of ROS production and cell wall modifications induced by the host-selective toxins ofPyrenophoratritici-repentisPtrToxA and PtrToxB Aakash Mankaney Ciuffetti Laboratory Department of Botany and Plant Pathology Oregon State University

2. Pyrenophera-triticirepentis (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. PtrToxA (ToxA) and PtrToxB (ToxB) • ToxA and ToxB are two proteinaceousHSTs isolated from P. tritici-repentis • ToxA and ToxB can be heterologously expressed in Escherichia coli and Pichiapastoris, respectively • ToxA necrosis (cell death) ToxBchlorosis (breakdown of chlorophyll) Ptr ToxA Ptr ToxB Sensitive Insensitive

5. D R G Mode-of-action of PtrToxA • Internalizes in ToxAsensitive mesophyllcells • 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 PtrToxBwill 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. 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. 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. MelaniaBetts Dr. IovannaPandelova Viola Manning