What are SA (salicylic acid) analogs? What is the structure of SA and their analogs?
INA
BTH
synthesized for commercial purposes
SQ: How can you explain the outcome of the experiment shown on Slide 12 (SA pretreatment, PAL expression)? How could you obtain evidence for the hypothesis (Pre-treatment with SA increases immune response)?
In th experiment, a northern blot was conducted to detect PAL mRNA (=Phenylalanine ammonia-lyase (marker enzyme for plant antimicrobial reaction)
Outcome experiment:
plant didn’t express PAL without PAMP = negative control
after presence of PAMP: PAL expression, stronger with increasing pre-treatment with SA
SA induces signaling components leading to more effiecient PAMP- or R-gene mediated signaling (doesn’t know exactly where SA works in this process)
Hypothesis proof: Northern blot, qRT-PCR
What does “N” stand for? How are plant cultivars called “nn” and “NN” different?
N is an immune receptor in plants. It is an R-protein. Plants with NN have the immune receptor, which recognize pathogen attack and trigger HR, causing immunity. Plants with nn don’t have the immune receptor and are therefore succeptible.
What is SAR? How can it be shown experimentally?
system acquired resistance = SAR
Experiment
1. step: infection with TMV of a lower leaf
2. step: infection of upper leaf after few days
result: SAR: shown if upper leaf has smaller HR lesions
priming of plant immune system is also possible with spraying SA instead of 1st infection
What happens during the first infection stage of SAR?
PR (pathogenesis-related) gene expression is induced after
TMV infections and SA treatment
PR proteins are markers for a general inducible defense program that is efficient against a broad range of pathogens( e.g. antifungal) (no antiviral proteins)
Is systemic acquired resistance pathogen specific?
No
In Arabidopsis: 1st infection with pseudomonas syringae pv. tomato avrRpt2
2nd infection with powdery mildew (Hyaloperonospora arabidopsidis) —> SAR
What is NahG/ what does the nahG mutation cause?
NahG = salicylate hydroxylase
naturally doesn’t occur in arabidopsis
used to produce SA mutants
mutants can’t accumulate SA —> prevents SAR
SQ: How can you isolate mutants deficient in SA biosynthesis? Name examples for mutants which are deficient in SA biosynthesis
Forward genetics:
mutagenesis of seeds
important to mutate both stem cells of shoot apical meristem
e.g. with chemicals
homozygous mutation necessary
SA deficient mutants
e.g. eds16 (enhanced disease susceptibility phenotype) no SAR (few SA procution, no PR1 transcripts)
e.g. sid2 (salicylic acid induction deficient)
eds16& sid2 are allelic (=exactly in the same gene)
eds5 transporter in chloroplast inner membrane: usually transports Isochorismate from chloroplast to cytosol (in cytosol only reaction to SA)
pbs3
eds5
Which SA biosynthesis do you know?
(SQ: For a long time, it was assumed that SA is synthesized from the amino acid phenylalanine. How was the alternative pathway discovered?)
2 Pathways for SA production —> ICS (Isochorismate) and PAL (Phenylalanine ammonia lyase)
pathway of SA synthesis via isochorismate was known in bacteria: chorsimate pathway
later also discovered in plant chloroplast —> bacterial origin
pbs3 enzyme discovered through mutant screens (SA deficient)
What is the ICS synthesis pathways of SA? (SQ: What does pbs3 do?)
In chloroplast: chorismate is converted to isochorismate (enzyme: Isochorismate synthase (ICS)) —> transported with EDS5 to cytoplasm
In cytoplasm:
PBS3 is a cytosolic enzyme that forms SA amino acid conjugates
conjugates are inactive forms
in pbs3 mutants, SA and amino acid conjugates are lacking
What are the three layers of the immune system?
In which layer of the immune system does SA play a role?
SA only marginally contributes to PTI
SA is necessary to limit bacterial growth in the context of ETS
SA is necessary for ETI
experiments that prove this: count colony forming units (cfu) of pseudomonas in WT (Col-0) and sid2 mutant (SA deficient)
What were the experiments to hunt for genes involved in SA signal transduction
2 Groups: Ryals and Dong
in nim1 mutant —>PR genes are not induced after INA oder SA
GUS reporter system only works in presence of SA (blue staining only when SA is there)
SAR not established in npr1 mutant (nno blue staining)
What is Npr1?
non expressor of PR
NPR1 is a receptor for SA
How was is discovered that NPR1 is the signal receptor AND signal transductor?
Required for signaling transduction:
SAR is not established in npr1 mutants
Also a receptor:
Experiment with radiolabeled SA —> size exclusion chromatography to purify NPR1 w/ bound labeled SA —> NPR1 binds to salicylic acid
NPR1 has a binding pocket for SA (Arginin), this amino acid was mutated to a different one (Glu) —> npr1 lines were complemented with normal NPR1 and mutated AA NPR1 (no SA bindin site) —> binding capacity is important for NPR1 function
WRKY70 is a gene that is induced by SA. Gene is not expressed in npr1 mutant.
Plants with mutated NPR1 could not express WRKY70
SQ: Imagine that you have isolated a mutant that does not produce SA. However, another group has already published a mutant with the same phenotype. Imagine that you cannot do a PCR because of shortage of Taq Polymerase. Can you imagine a way to find out whether your mutation is in the same gene that was published before?
Genetic inheritance of mutation genes are usually recessive.
Crossing experiments of 2 discovered mutant strains
—> if the mutations were in the same gene, the inability to produce SA stays in tact
—> if the mutation was in different genes, phenotype of cross is back to wt (can produce SA)
SQ: Design an experiment that answers the question whether NPR1 has to move to the nucleus to activate gene expression.
GPF fusion of NLS (nuclear localisation sequence), fluorescence microscopy of nucleus (see if NPR1 in nucleus)
What is TGA? Which TGA factors are essential for SA signaling?
TGA transcription factors belong to the superfamily of bZIP transcription factors. TGACG is an essential cis element of the PR1 promoter.
TGA2, 5 and 6 are essential for SA signaling.
The tga256 is deficient in SAR establishment.
SQ: How would you design an experiment to find out, whether the TGACG motif is important for PR1 promoter activity?
Mutate the anticipated target of PR1 (TGACG motif) and see if expression of PR1 is impaired (for example with GUS reporter)
How was ist experimentally found out that NPR1 interacts with TGA?
Yeast two hybrid system (Y2H)
In Yeast: bait protein can “fish out” prey (prey can be unknow or already known)
BD = DNA binding domain
AD = activation domain —> activates RNAPol II —> expression of reporter gene
activation of RNAPol II only when bait and prey in close proximity = only when 2 proteins interact
Which types of TGA proteins are necessary for SAR establishment?
TGA2, TGA 5, TGA6
Old SQ: You have learned in the lecture that SA is not the mobile signal. How could grafting experiments (wt and nahG plants) help to provide evidence for this hypothesis?
Graft two plants together: Lower part ––> nahG; Upper part ––> WT
If SAR is still induced in the upper leaves after infection of the lower leaves, SA cannot be the mobile signal since nahG degraded SA
SQ: Describe two experiments (other than Y2H) to confirm that TGA proteins and NPR1 interact in planta
Immunoprecipitation assay, FRET, (PR1 expression experiment with mutated TGACG sequence and GUS reporter)
SQ: NPR1 was found in a screen using the PR2 promoter. The PR2 promoter does not contain TGA binding sites. How can you explain that the PR2 promoter is not active in the npr1 mutant?
NPR1 and PR2 are connected via an indirect mechanism.
NPR1 binds to TGA and activates the expression of WRKY, another transcription factor. WRKY is a positive regulator for PR2 expression.
in npr1, NPR1 is missing, so the cascade for producing PR2 is not activated
SQ: Posttranscriptional control mechanisms can involve the reduction of disulfide bridges. Which protein do you know that is subject to such a modification? Which process is influenced by this modification?
NPR1 exists as an inactive oligomer in the cytosol
Disulfide bonds are reduced to release monomeric NPR1, upon SA formation in cell.
Thioredoxin h5: TRXh5
TRXh5 transcription is induced by SA-activated NPR1. The trxh5 mutant is (partially) deficient in SA-induced PR1 expression and SAR.
What are thioredoxins and what do they do?
TRXh5 in arabidopsis
transfer electrons to target protein (in this case NPR1)
SQ: One allele of npr1 was isolated by screening for Arabidopsis mutants that did not develop resistance against biotrophic pathogens after treatment with INA. Describe the experimental approach how it can be tested whether the mutant is also deficient in “systemic acquired resistance”.
Do “classical SAR experiment”: Lower leaf and later upper leaf infection with Psm, count cfu
Give a simple overview of SA signaling
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