Practical questions

1. Prepare standards: pipet 0,3,5,7,10,15 µl of a 1mg/ml BSA solution —> read abosprtion at 595 nm

2. Read absorption at 595 nm of your protein samples with added Bradford mix

3. create standard curve (y axis: A595, x axis: µg BSA)

4. Read measured absorption of your sample on graph (corresponding x axis value = protein amount µg) and divide by volume used to get final protein concentration (µg/µl)

Expected results:

Total extract CERK1

• WT: should show a band + band shift —> CERK1 is present (and phosphorylated after PAMP treatment)

• receptor mutant cerk1: no band +no band shift —> no receptor present, no phosphorylation

• co-receptor mutant lyk4/lyk5: band + no band shift —> CERK1 present but no phsophorylation because no co-receptors

Chitin pulldown CERK1

• WT: band + band shift (possibly weaker in +PAMP because of saturation)

• receptor mutant cerk1: no band + no band shift

• co-receptor mutant lyk4/lyk5: band + no band shift —> ligand is bound by receptor and co-receptor together (molecular glue model) —> without co-receptor, band might be weaker

Anti MAPK

only PAMP treated WT should have a band, mutants can’t start MAPK signaling

• Only WT should show chemiluminescence and therefore reaction to PAMP treatment

• receptor and co-receptor are both needed to recognize PAMP and therefore to activate RBOHD (NADPH oxidase) and produce ROS

• recap activation of RBOHD: PAMP perception by receptor —> phosphorylation co-receptor —> phosphorylation RLCK BIK1 —> phosphorylation + activation RBOHD —> ROS production

Incompatible (no disease, no cfu): Psm 4326 avrRpt2 + Col-0 (R gene + avr protein) —> resistant plant, avirulent pathogen

Compatible (=disease, high number cfu):

• Psm4326 + Col-0 (Psm has no avirulence protein)

• Psm4326 avrRpt2 + Col-0 rps2-101 (arabidopsis has no effector gene)

• Psm4326 + Col-0 rps2-101 (Psm no avirulence gene, arabidopsis no R-gene)

—> susceptible plant, virulent pathogen

Increasing electrolyte leakage in incompatible reactions since this is associated with cell death (HR!—> no disease)

No electrolyte leakage for compatible interactions (no HR, disease)

1. normalize conductivity by dividing measured conductivity by conductivity value after autoclaving (=total amounts of electrolytes in sample)

2. substract normalized conducitivity values for Psm infected plants with values of mock control —> relative conductivity

• Light energy absorbed by chlorophyll molecules can undergo 3 fates:

  • used for photosynthesis (photochemistry)

  • excess energy dissipated as heat

  • re-emission as light

• any increase in efficiency of one will result in decrease of the other two

• This means that change in fluorescence yield (reemission of light) —> change in photochemical efficiency and heat dissipation

• Fo ground state value induced by measuring light

• Saturation pulse (SP): fluoresence reaches maximum level (Fm) —> primary electron acceptor of FSII (=QA) is fully reduced —> pulse too short to induce dark reaction

• actinic light (AL) = constant illumination induces photochemistry —> rise because of lag phase before carbon fixation

• Saturation flashes (SP) while AL active —> Fm’ is lower than Fm, because QA again fully reduced

Lower maximum quantum efficiency (Fv/Fm) ––> lower photochemistry ––> less photosynthesis due to dead cells (HR) ––> incompatible reaction

Therefore:

Lower Fv/Fm = HR = incompatible reaction

Lower Y(II) = HR = incompatible reaction

Higher qN = HR = incompatible reaction

• incompatible reaction: lower photochemistry reaction —> photosystem is impaired due to HR

  • lower maximum quantum efficiency and quantum yield

    
    

• the TMV helicase

• W38 —> No N gene (=R gene) —> disease, susceptible (visible after 6 days of infection)

• Xanthi (N gene = R gene) —> HR lesions, resistant

Mock: bigger HR lesions on upper leaf —> no SAR because no primary infection

TMV: smaller HR lesions —> SAR because primed with pramary infection

Col-0: mock and psm treated: PR1 expression (higher in psm treated!) —> SAR establishment in psm treated

npr1-1 mutant: no PR1 expression (neither in mock or psm treated) —> no SAR

tga256 mutant: no PR1 expression (neither in mock or psm) —> no SAR

• negative control (=H2O+primer) should have no signal/curve

• melting curves —> only 1 peak (specific product has its specific peak. If 2 peaks or more= another product present or primers homodimerized/formed hairpin loop)

• threshold defined in the exponential phase to receive CT values (logarithmic style of graph required)

• Check Ct values —> everything above Ct=38 —> non-detectable

• reference gene in this case UB5, but can also be e.g. actin, tubulin etc.

Relative expression:

Set untreated WT to “1” —> mean of the fold over reference (for) is always needed

Divide the given fold over reference by the mean of untreated WT sample

Example:

WT1 for=0.68; WT2 for=0.22 —> mean=0.45

sample for=0.09 —> relative expression=0.09/0.45=0.2

eukaryotic: 25S, 18S, 5.8S, 5S rRNA + cleaved prokaryotic 23S rRNA (chloroplasts)

• reporter gene system

• GUS enzyme catalyzes the hydrolysis of beta-glycosidic bonds in glucronidase —> results in a coloured product

• Purpose: can answer whether the transgene is expressed within the plant and under which conditions the promoter is active + how strong the expression is

• allows in situ localization —> analysis of tissue specificity of promoter

• Calculate Δfluorescence (Ft=60 – Ft=0)

• Calculate the MUG turnover in pmol

• Calculate how many pmol are in one fluorescence unit

  • 1 µM = 1 pmol/µl

    200 µl aka 200 pmol of MU of each dilution had been added to the plate ––> 1 fluorescence unit = 200/169 (the 169 was a value from the dilution series of the MU standard (3.4.))

  • MUG turnover = fluorescence unit x Δfluorescence (How much

• Calculate the MUG turnover per time (GUS unity)

                          t = 60 min

                          MUG turnover per time = MUG turnover/t

• Calculate MUG turnover per time and total protein (spec. GUS activity)

  Total protein = 12.5 µg (in the beginning, 25 µg were added and then divided into two reactions)

  MUG turnover per time and total protein = MUG turnover per time/12.5 µg

   

• method to study protein-protein interactions in vivo

• heterlogous expression of hybrid fusion proteins in S. cerevisiae (in nucleus)

• bait (known) is fused to DNA binding domain (DBD) = yeast transcription factor GAL4

• prey (unknown) is fused to activation domain (AD) = GAL4 transactivation domain

• upon interaction of prey and bait —> RNAPol II activation, transcription of reporter gene

• electrophoretic mobility shift assay

• determine DNA/RNA-protein interaction

• electrophoretic separation of RNA/DNA-protein mixture on polyacrylamide or agarose gel

• control lane = DNA or RNA without proteins

• band shift indicates protein-DNA interaction (bigger size than only DNA or non-interacting DNA)

• proteins need to be transported into nucleus (NLS required)

• membrane proteins therefore can’t be studied

(-TRP-LEU) —> confirmation if transformation worked

(-TRP-LEU-ADE-HIS) —> confirmation if there was protein interaction

On the plasmids, genes which can produce amino acids (TRP, LEU, etc.) . Reporter genes GAL1/2 of used yeast strain (PJ69-4A) are connected with genes which can prodcue ADE and HIS.

• NPR1 in combination with TGA2/TGA6 leads to growth on plates (-TRP-LEU-ADE-HIS) = INTERACTION

• NPR in combination with TGA1 —> no growth on (-TRP-LEU-ADE-HIS) = no INTERACTION

• npr1-1 with any combination —> no growth on (-TRP-LEU-ADE-HIS) = no INTERACTION

• detection of enzyme beta-galactosidase

• yeast strain contains lacZ gene (from E.coli) —> encodes beta-galactosidase (controlled by GAL4 promoter)

• cell lysis of cells containing expression plasmid —> mixing with artificial beta-galactosidase substrate o-nitrophenyl-β-D-galactopyranoside (ONPG, colorless)

• cleavage of ONPG by reporter enzyme —> galactosidase + o-nitrophenol (ONP, yellow colour)

  • only works if fusion of proteins bait and prey and expression of GAL4 factor (=beta galactosidase expression)

• yellow colour allows colorimetric quantification (420nm—> OD420)

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• colorimetric quantification (420nm—> OD420)

• normalization with OD600 = number of cells

• the stronger the interaction between NPR1 and TGA, the higher the beta-Gal units

• many pathogen-inducible plant promoters (e.g. PR-1) have regulatory sequences related to the activating sequence-1 (as-1)

• promoters are activated by signal transduction pathway which requires SA

• exact mechanism of as-1 dependent gene regulation is unknown (maybe DNA binding of postive-acting TFs is regulated and occurs only in presence of SA)

• as-1 elements have TGACG sequence motifs which are recognized by dimers of TGA factors

• protoplast generation (enzymatic solution hydrolyzing the cell wall) of sid2 and sid2npr1 plants

• artificial promoter (contains as-1 element) is fused to the reporter gene fLUC (encoding firefly luciferase) —> plasmid as-1:fLUC

• when additional second plasmid is transformed, which codes for a TF (effector plasmid) the influence of this protein on the expression of the reporter gene can be dissected

• effector plasmid: TGA2 TF and a strong constitutive promoter (VP). fusion to HA = negative control (doesn’t bind to DNA)

• measurement fLUC (firefly Luciferase) and rLUC (renilla luciferase) —> different fluorescence products

• direct gene transfer (PEG mediated)

• one day after PEG: determination of fLUC activity (measurement of luminescence

• fLUC= firefly Luciferase. fLUC is determined by measurement of luminescence

  • reaction: beetle luciferin —> Oxyluciferin

    • gives information about as-1 dependent promoter activity —> the better binding of TGA to as-1 element, the more fluoresence by the fLUC enzyme

• rLUC = renilla Luciferase. Reporter enzyme. Coding region on control plasmid is regulated by strong promoter UBQ10

  • reaction: Coelenterazine —> Coelenteramide

  • expression should be independent of external impacts

1. calculate fLUC/rLUC ratio of every sample

2. Mean of fLUC/rLUC ratio of negative controls —> set to 1

3. calculate relative ratio by dividing all fLUC/rLUC ratios by the mean on fLUC/rLUC of negative control

only in sid2 mutants, plants treated with SA —> TGA interacts with as-1 element and interaction increases from HA (only indigenous TGA) to TGA2 to TGA-VP (SA is required for induction of pathway and in sid2mpr1 mutant plants, NPR1 is missing —> signal can’t be passed to TGA).

Actual results were differently: only interaction between TGA2 and the as-1 element when TGA2-VP was present but then also without SA treatment and also in the sid2npr1 mutant plants. Why this happens is unclear. IT could be that SA receptors/transporters are located in cell wall and protplast have no cell wall —> different behaviour of protplasts (no SA detection in protoplasts)

• coomassie blue = fungal structures

• aniline blue = callose deposition

• WT: callose accumulation at sides of fungal invasion

• pmr4 (encodes gsl5 callose synthase): no callose accumulation (synthesis is impaired)

• pen2 (encodes glycosyl hydrolase): callose accumulation, but still fungal penetration

Bgh is non-adapted (HR-like cell death induction —> incompatible reaction)

Go is adapted (compatible reaction, no HR induction) —> better penetration

• pen2 —> pre-inavsion defense is compromised —> glycoside hydrolase not functional, some toxic compounds can’t be produced

  only slight penetration in pmr4 (higher resistance although lacking of callose synthase)

pen2 > pmr4 > WT

• pen2 and WT. pmr4 most resistant

  
  

The green fluorescent protein (GFP) has an excitation wavelength of 488nm (blue light) and emits a spectrum of longer wavelengths (500-550nm) (green light)

• cellular polarization = common reaction of plant cells in response to pathogen attacks. polar rearrangement of cytoplasm and directional movement of various organelles (nucleus, golgi)

• pathways:

  • 1. pre-invasion resistance oathway (directed trafficking of vesicles with toxic compunds to site of invasion; mediated by SNARE protein PEN1)

  • 2. pre-inavsion resistance pathway ( relies on PEN2, associated with mitochondria and peroxisomes, formation of aggregates and penetration sites) PEN2 produces toxins, PEN3 (ABC transporter) transports it to fungal intruder

PEN-GFP fusion proteins focally accumulate at sites of attempted penetration

PEN1 and PEN3 are localized to the plasma membrane

PEN2 is associated with the mitochondrial and peroxisomal membrane

No transdifferentiation in Mock treated plants

Transdifferentiation in TRADE treated plants

bsc = bundle sheat cells (cells that surround xylem)

transdifferntiation of bsc = bsc turned into functional xylem vessels