1. Name four ROS.
Singlet oxygen
Superoxide anion
Hydrogen peroxide
Hydroxyl radicale
2. Draw a sketch which shows ROS generation during the first three hours after elicitation of PTI and ETI.
PAMP Triggered Immunity is first,
Then ROS production goes down as pathogen effectors supress PTI
Then plant reacts to Effectors during ETI and produces ROS again
Design an experiment with which you can induce only ETI (without PTI)
Arabidopsis Col-0 expresses the R protein RPS2
—> transformed with a construct encoding avrRPt2 (effector) under the control of a promoter which can be activated by estradiol (est). When spraying the plant with est, the CC-type NLR RPS2 is activated which induces ETI.
What is RBOH. Which type of RBOH is most important for ROS production?
RBOH= Respiratory Burst Oxidase Homolog. RBOH is a NADPH oxidase which transfers electrons to O2 to produce O2.- (superoxide anion). RBOHD is the most important one for ROS production (rbohd ko-mutant drastically reduces ROS production).
3. Why does the second wave of ROS generation during ETI not occur after infection by virulent pathogens?
During compatible interaction plant has R gene that recognizes Avr factor of pathogen and starts immune reaction. ROS is activated and no disease takes place.
For virulent strains, PAMPS are recognized and PTI is started, but the Effectors are not recognized. Enhanced disease is also caused due to the suppression of PTI by effectors.
How is RBOHD activated?
Upon PAMP recognition:
PRR recognize PAMPs (e.g. flg22/chitin)
via phosphorylation of BAK1 (co receptor of fls2) by FLS2, the complex is activated.
BAK1 transfers the phosphate to BIK1 (cytoplasmic kinase)
and therfore acitvates it
BIK1 mediates RBOHD phosphrylation and therfore activation
RBOHD oxides NADPH and transfers the electron to O2 to produce a superoxide anion (ROS)
via a superoxide dismutase, the superoxide anion is converted to H2O2
What is the requirement for activation of ETI and what are the effects of ETI activation regarding PTI?
To trigger ETI, PTI has to be activated first (PAMP & PRR —> leads to ROS production + upregulation of defence genes). ETI is then activated via recognition of effectors by NLRs. Activation of ETI leads to increased protein production of proteins involved in the PTI signaling. This leads to a much more effective immune response.
4. ROS accumulation interferes with fungal growth during the maize / Ustillago maydis interaction. How might plant generated ROS interfere with fungal growth?
Barrier fortification: cross linking of pectin, ROS increases Callose deposition at cell walls —> harder for pathogen to penetrate
Direct toxic effect of ROS on pathogens
Modification of protein activitiy (aquaporin usually transports H2O, but can also transport H2O2)
How can you explain the direct toxic effect of H2O2?
Fenton reaction generated hydroxyl radicals
The radicals lead to rapid lipid peroxidation —> propagation of radicals (radikal klaut immer elektronen, die das molekül gar nicht abgeben wollte, und erstellt dadurch nächstes radikal)
Lipid peroxidation leads to reactive electrophilic species (RES)
RES bind to proteins at sulfhydryl and amino groups
Fenton reaction:
How are PTI, ETI and SAR dependent/not dependent on RBOHD (NADPH oxidase)?
PTI: independent (acitvates RBOHD)
ETI: dependent on RBOHD —> RBOHD contributes to activation of ETI, programmed cell death
SAR: partially dependent
5. Describe two peroxidase-catalyzed reactions that lead to cross-linking of cell walls.
(Tutorial: Dont draw structures but keep in mind that radicals are involved)
Detrosin (expansin cross linking)
cross-linking of proteins through dityrosine:
Cross-linking of pectin through ferulic acid
6. Name four types of post-translational modifications of reactive SH groups under conditions of oxidative stress.
Sulfenic acid, then modification
7. H2O2 is discussed to be a signaling molecule. This has been shown in yeast and other fungi like Ustillago maydis. Describe the signal transduction pathway leading to the activation of gene expression under increased H2O2 conditions.
Under Non-stressed conditions: Yap1 Import and mainly export out of nucleus
Under oxidative stress: Yap1 import in nucleus, almost no export
In cytoplasm, Gpx3 (glutathione oxidase) reacts with H2O2 leading to SOH group at Gpx3 —> Gpx3 binding to Yap1 via disulfide bridge under release of water —> Gpx3 oxidizes SH-group of Yap1 -> leads to disulfide bond at NES sequence —> import to nucleus, no export of Yap1 out of nucleus anymore (NES is blocked by disulfide bridge)
Very rare example that a protein is regulated in its function by sulfidation
8. What is lipid peroxidation?
propagation of radicals (radikal klaut immer elektronen, die das molekül gar nicht abgeben wollte, und erstellt dadurch nächstes radikal)
unsaturated lipid —> radical (produced in Fenton reaction) initiates oxidation (by extraction of H) —> lipid radical can react with O2 —> peroxidation
9. How do radicals initiate the oxidation of biomolecules?
Via cleavage of one proton
10. Describe how ascorbate can terminate the chain reaction initiated by radicals.
Ascorbate becomes the radical, then either 2 ascobates dimerize spontaneously, or dimerisation is catalized by monodehydroascorbate reductase under NADH usage
11. What is glutathione?
An antioxidant with gamma peptide linkage (cant be cleaved by peptidases)
Is required for regeneration of reduces ascorbate (Glutathion reductase)
Can scavenge H2O2 for neutralisation (creates H2O and dimerised glutathion) (Glutation peroxidase)
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