Sample preparation for monosaccharide analysis using HPLC Sample preparation for polysaccharide analysis using carbohy-

drate gel electrophoresis (PACE)

Reagents
1. Millipore water Reagents
2. 15% trifluoroacetic acid (TFA) 4 M NaOH
3. 0.5 M 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP) in methanol 1 M HCl
4. 0.5 M NaOH
100 mM Ammonium acetate pH 5.5
5. 0.5 M HCI
6. Chloroform Glycoside Hydrolase (enzyme)
Protocol 8-Aminonaphthalene-1,3,6-trisulfonic acid (ANTS)
1. Dissolve 4 mg dried cell wall samples in 1 mL Millipore water. 6 M Urea
2.Aliquot 250 µl solution into screw-top Eppendorf tube to obtain Protocol
1 mg sample 1.Weigh 1 mg AIR dried sample (biomass) into 1.5 mL Eppendorf
3. Dry the solution in miVac centrifugal concentrator at 35 °C for
tube.
2 hours.
2.Alkali extraction – Add 20 μL 4 M NaOH to the biomass and ex-
4. Add 500 µl of 15% trifluoroacetic acid (TFA).
tract for 1 hour at room temperature (for more biomass it may be nec-
5. Heat at 120 °C for 2 hours.
essary to use more NaOH the clue is to wet all AIR).
6. Carefully pick with forceps, chill on ice, Wear safety goggles (2-
5Minutes) 3.Neutralization – Add 80 μL (~83 μL to cater for leakage and evapo-
7. Spin for 10 min at maximum speed. ration) 1 M HCl and add 900 μL 0.1 M (100 mM) ammonium acetate
8. Transfer 400 µL of supernatant to 2 m. Eppendorf safe-lock tube. pH 5.5 [This may vary between enzymes]. Mix thoroughly by pipet-
9. Dry in Nitrogen gas for 2 hours or overnight. ting. At this stage samples are stable in -20 up to a year.
10. Add 25 µL 0.5 M 3-methyl-1-phenyl-2-pyrazoline-5-one [PMP) 4.Digestion – Digest about 100 μL (100 μg) with Glycoside Hydrolase
to the dry biomass and miss thoroughly by pipetting. It is light sensi- (enzyme) and incubate at 30 – 37 °C (according to the enzyme’s activ-
tive, so keep away from direct light. ity) for preferably 16 – 18 hours with regular shaking at 1000 rpm.
11. Add 15 µL 0.5 M NaOH. Before digestion, vortex the extracted sample and digest desired
12. Add 10 µl Millipore water and mix gently.. amount WITH sediment! Do not take only supernatant. If the sedi-
13. incubate at 70 °C for 2 hours. Cover with aluminum foil, prefera- ment blocks the tip, cut it with scissors.
bly. 5.Lyophilization – Lyophilize in miVac centrifugal concentrator at 37
14. Carefully pick with forceps, and chill on ice for about 5 min. °C for 2 hours in the dark. Cover with aluminum foil, preferably.
Wear safety goggles. 6.Derivatization – Add 6 μL 8-Aminonaphthalene-1,3,6-trisulfonic
15. Spin at max speed for 1 min.
acid (ANTS). The volume of ANTS can be increased if you have
16. Neutralize with 20 µL 0.5 M HCI.
more biomass. Mix gently by pipetting, followed by a quick spin and
17. Wash with 700 µl chloroform. Perform this process in fume
incubate overnight at 37 °C in the dark.
hood. This process removes unbound and excess PMP on the sugars
7.Lyophilization – Lyophilize in miVac centrifugal concentrator at 37
to ensure effective separation by the HPLC system.
18. Vortex for about 30 sec and spin at about 2000 rpm for 1 min. °C for 30 min in the dark. Cover with aluminum foil.
There will be phase separation 8.Cast PACE gel and set up the apparatus for carbohydrate electro-
19. Transfer the water phase (aqueous layer) to new tube. Discard phoresis (refer to next page).
organic layer. 9.Resuspend ANTS labelled samples in 25 μL 6 M urea for loading
20. Wash the water phase with 700 µL chloroform. Perform in fume (Refer to next page). [If you don’t want to run PACE immediately, do
hood. not resuspend in urea, rather freeze samples in -20. Samples are
21. Vortex for about 30 sec and spin at about 2000 rpm for 1 min. stable in -20 for ~2 months].
There will be phase separation.
22.Transfer the aqueous layer to new tube, discard the organic layer Polysaccharide analysis using carbohydrate gel electropho-
23. Dry the water phase in miVac at 35 C for 1 hour. Cover the lid of resis (PACE) setup
the mivac with aluminum foil to prevent light. Reagents/Chemicals (10 % gel), Distilled water, 1 M Tris-
24. Store the dried sample at -80 °C (for up to two weeks) for subse- borate buffer, Acrylamide 29:1, 10 % ammonium persulfate
quent HPLC analysis. (APS), Tetramethylethylenediamine (TEMED)
Method/Protocol
1. Place together glass plates, spacers, and holders. Do not
screw to tighten because glass is prone to cracking. Ensure
that the spacers are placed straight and aligned to the glass
edge to avoid any leakage. Place the prepared glass tightly
onto the seal in the rack and screw.
2. To make 10 % gel, mix in 50 mL falcon tube 23.3 mL
distilled water, 3.5 mL 1 M Tris-borate buffer and 9.8 mL
Acrylamide 29:1 (at this point, the mixture can be stored
in the refrigerator for future use). Before casting add 210
μL 10 % APS and 28 μL TEMED (under a fume hood).
Mix well with shaking or vortexing.
3. Use a 5 mL pipette to cast the gel by gently filling the
space between the glass plates to the brim with the gel
solution, using a 5 mL pipette. Ensure there’s no air bub-
bles, and insert the comb. If there’s any leakage, add more
gel solution until it solidifies.
1. Fungal EPS Inoculation experiment 4. Allow the gel to stand for approximately 20 min to polymer-
ized. To avoid drying, cover the top of the gel (the glass
a. Divide plant samples into three groups slides) with wet tissue paper. Subsequently, cover the entire
I. Treat group one with EPS from H. fraxineus. apparatus with a plastic bag and keep in the cold room over-
night.
a. Determine the concentration of EPS before use 5. Resuspend ANTS labelled samples in 25 μL 6 M urea
i. Measure OD600 of EPS solution (typically 25 μL 6 M urea for 100 μg digested AIR) for load-
ing.
ii. Calculate amount of EPS [in ppm] in EPS solu-
6. Remove the gel holder (with the glass plates, spacers and
tion (Santra and Banerjee et al 2022; https://
holder) from the rack and place it in an electrophoresis cas-
doi.org/10.3389/ffunb.2021.796010). Note that 1
ppm is equivalent to 1 mg of solute per liter of solu-
sette.
tion. 7. Mark the wells with marker, leaving the wells on both ex-
treme ends.
II. Treat group two with Millipore water 8. Place the cassette into the tank and fill the cassette up to the
III. Treat group three with 50 uM MLG43, as positive electrode (wire in the black plastic element) with 0.1 M Tris-
control. borate pH 8.2. Fill the tank up to the bottom glass edge. If
there is already a buffer inside, there is no need to add an
additional buffer to the tank.
2. Gene expression studies (RT-qPCR) 9. Wait a bit to see if the buffer level does not change; if so,
there is a leakage. If so, screw the holders once again or
a. Harvest 2-3 leaf samples for gene expression studies.
monitor the buffer level during the run and add more buffer
Immediately store sample in liquid nitrogen prior to use or
at -80 degree for future use.
if needed.
10. Load ~5 μL of the standard and ~3.9 μL of the sample into
i. 30 min after treatment [Water, EPS and MLG43] wells, respectively. If possible, avoid wells on both extreme
ii. 60 min after treatment [Water, EPS and MLG43] ends, as usually migration of samples tends to bend.
11. Place the lid (there is only one possibility to fit the lid) and
b. Extract RNA from samples using available kits, and cover the tank with dark material to avoid light.
quantify using nanodrop 12. Start the run. Turn on the power pack, choose the manual,
c. Synthesize first strand cDNA and enter 500 V. Push “RUN”. Run for about 50 min. Using
a freshly prepared buffer, the current strength will be ap-
d. Perform RT-qPCR using selected genes
proximately 31 mA, but may vary as the buffer is reused. A
i. Disease response genes reused buffer usually provides straighter PACE bands.
13. Monitor the current strength and buffer level during the run.
1. WRKY30 and WRKY33
Whenever you take off the black material, STOP run. Other-
2. CNCG4 and CML37 wise, the samples will interfere with acrylamide, and you
ii. Plant defensin genes (https://www.tandfonline.com/ may get additional marks on the gels.
doi/full/10.4161/psb.4.11.9755) 14. When the electrophoresis time is over, push STOP on the
power pack.
1. Members of plant defensin (PDF) family include 15. Take the cassette out and pour the buffer inside the tank. Pull
AT5G44420 https://www.arabidopsis.org/locus? out the gel holder (with the glass plates, spacers and holder)
key=133224) from the cassette. Cover the gel holder with black material to
avoid direct exposure to light. Transfer the gel holder for
imaging.
3. Identify signal pathway simulated by elicitors using
16. You can image the whole gel holder without removing the
histochemical GUS assay (Venegas-Molina et al. 2020;
https://doi.org/10.1038/s41598-020-67074-7).
gel from glass to check if electrophoresis is complete. Use
low exposure time. If the gel needs additional run time, place
a. PATHOGENESIS-RELATED GENE 1 (PR1) reporter it the cassette and run electrophoresis. Wash the transillumi-
constructs, as defense marker gene involved in salicyclic nator with water. Water can be sprayed onto a transillumina-
acid (SA) signaling pathway tor to avoid gel drying and to achieve better gel placement.
b. LIPOXYGENASE 2 (LOX2) reporter constructs, as 17. If the run is ready, unscrew the holders and separate the
defense marker gene involved in jasmonic acid (JA) signal- glass with the western blot green tool. Gently transfer the gel
ing pathway to the transilluminator holding the bottom edge, avoid air
bubbles.
c. PG15::GUS reporter construct as a control for constitu-
18. Image the gel in UV transilluminator.
tive GUS expression gene