Elucidating #946;-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

The study was conducted with the approval and permission of the Environmental and Biosafety Research Ethics Committee of the University of the Free State (UFS-ESD2022/0131/22). The details of the reagents and the equipment here are listed in the Table of Materials.

1. Plant growth conditions

1. Germinate 250 seeds of each wheat cultivar, i.e., susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5, in separate Petri dishes.
2. Add 5 mL of distilled water in each Petri dish, seal with a paraffin film, and incubate in the germination chamber set at 25 °C for 2 days.
3. Transplant the germinated seeds of each cultivar in 15 cm pots containing 1:1 soil and peat moss (15 plants per pot) under controlled greenhouse conditions.
4. Set the temperature regimes at 18 °C and 24 °C during night and day, respectively.
5. Irrigate the plants every 3 days using tap water and supply them with 2 g/L fertilizer 14 days after germination.
6. Place the plants in cages encased in nets and allow them to grow to the third leaf stage^2,6 (with four biological replicates of each cultivar).

2. Wheat cultivars infestation with RWASA2

1. Infest Tugela, Tugela-Dn1, and Tugela-Dn5 wheat cultivars with RWASA2 according to Jimoh et al.¹¹ and Mohase and Taiwe¹².
2. Keep the plants in two separate sets in cages; infest one set with 20 parthenogenetic individuals of the same RWASA2 clone per plant in each pot in the growth chambers covered with nets. Keep another set of wheat plants in a separate room under the growth chamber with clean nets and treat them as controls.
   NOTE: Keep the two treatments in separate rooms and cover the plants in cages encased in nets. Also, in a case where two different RWA biotypes are used in a study, keep the plants infested with one RWA biotype from those infested with the second biotype as far as possible or in separate rooms under growth chambers covered with nets to avoid any possible cross-contamination of the RWASA2.
3. For harvesting, select the second and third leaves at two different time regimes, short-term feeding period (1, 2, and 3 days) and long-term feeding period (7 and 14 days), and wrap them in moist paper towels.
4. Immediately transfer the harvested leaves to a box containing ice to reduce leaf metabolisms before intercellular wash fluid (IWF) extraction.

3. Extraction of intercellular wash fluid (IWF) from the apoplast

1. Cut the harvested leaves of about three wheat cultivars into 7 cm pieces.
2. Rinse the leaf pieces twice in distilled water to remove any cytosolic contamination from the cut ends^6,13.
3. Insert the leaf pieces into a thick-walled glass tube and submerge the samples in extraction buffer (50 mM Tris-HCl, pH 7.8).
4. Apply vacuum infiltration for 5 min using a water jet pump to impregnate leaves with the extraction buffer.
5. Thereafter, remove the leaf pieces from the glass tube and blot dry them with a paper towel.
6. Insert the dried leaf pieces vertically into pre-cooled centrifuge tubes fitted with perforated disks and centrifuge at 500 x g for 10 min at 4 °C.
7. Use a 100 µL pipette to collect the supernatant into pre-cooled 1.5 mL microcentrifuge tubes.
8. Repeat the entire extraction procedure with the same leaf material.
9. Combine the collected supernatant and store it at −20° C.
   NOTE: The supernatant aliquots are treated as the source of apoplast content or IWF. The IWF was not used in the current study, but it was important to remove it from the leaf tissues before we extracted the cell wall-bound β-1,3-glucanase and Peroxidase (POD).

4. Extraction of the cell wall-associated β-1,3-glucanase and POD

NOTE: The leaf residues left after IWF extraction were used to extract total cell wall protein.

1. Crush the leaf residues to a fine powder with liquid nitrogen using a mortar and pestle.
2. Transfer approximately 200 mg of the powdered leaf tissue into a mortar containing 4 mL of extraction buffer (50 mM sodium phosphate with 1% (w/v) polyvinylpolypyrrolidone (PVPP); pH 5.0) followed by grinding with a pestle to a smooth paste, which is then transferred to microcentrifuge tubes.
3. Incubate the mixture in the microcentrifuge tubes for 3 min on ice and then centrifuge at 10,000 x g for 15 min at 4 °C.
4. Collect the supernatant (total protein extracts) in microcentrifuge tubes (aliquots of 2 mL) and use it as a source of β-1,3-glucanase and POD for the entire study.

5. Determination of the protein standard

1. Determine the total protein concentration of the extracts following the Bradford method¹⁴.
2. Prepare the bovine serum albumin (BSA) solutions at the concentrations of 0.0 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL and 1.0 mg/mL dissolved in 50 mM sodium phosphate buffer to generate the protein standard.
3. Prepare the standard by adding 10 µL of each BSA solution into the 96-well microplate with 190 µL (0.25% v/v) of Bradford reagent (see Table of Materials).
4. Incubate at 25 °C for 20 min and measure the absorbance at 595 nm using the microplate reader and use the absorbance values to prepare the standard curve for quantifying protein concentration.
5. Quantify the protein concentration
   1. Add 10 µL of the extracted enzyme (step 4) into a 96-well microplate with 190 µL (0.25% v/v) of Bradford reagent in triplicates.
   2. Incubate at room temperature (25 °C) for 20 min.
   3. Measure the absorbance at 595 nm using the spectrophotometer with a microplate reader and use the absorbance values to determine the protein concentration using BSA as the protein standard (prepared in step 5.4).

6. Prepare glucose standard for β-1,3-glucanase activity

1. Prepare the standard solutions with concentrations between 0.00 mg/mL and 1.0 mg/mL (0.0 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL) with glucose dissolved in distilled water.
2. Add 300 µL of each glucose solution and 600 µL of 3,5-Dinitrosalicylic acid (DNS) reagent at a 1:2 ratio into 1.5 mL microcentrifuge tubes and incubate the reactions at 100 °C for 5 min (see Table of Materials for DNS reagents) (Miller et al.¹⁵).
3. Allow the mixture to cool at room temperature before measuring the absorbance at 540 nm using the spectrophotometer.
4. Use the average absorbance values to develop the standard curve for determining β-1,3-glucanase enzyme activity.

7. Determining β-1,3-glucanase activity assay

NOTE: The enzyme activity of the cell wall-bound β-1,3-glucanase extracted from Tugela, Tugela-Dn1, and Tugela-Dn5 was determined by the amount of released total reducing sugars released from hydrolysis of 0.5% (w/v) mixed linked β-1,3-1,4-glucan (MLG) and 0.4% (w/v) β-1,3-glucan substrates using a modified method as described by Miller et al.¹⁵. Always keep the tubes with protein aliquots on the ice. If the samples were frozen, thaw them on ice and proceed immediately after thawing.

1. Add 200 µL of the enzyme extract (keep the protein concentration between 20 µg/mL to 90 µg/mL in all reactions, unless stated otherwise) and 300 µL of the substrate dissolved in 50 mM sodium citrate buffer (pH 5.0) into the 1.5 mL microcentrifuge tubes.
   NOTE: Run a parallel blank reaction without the enzyme extract for all substrates (first blank), and the enzyme blank consists of enzyme extract without substrate (second blank). Both the substrate and enzyme extract were replaced with the buffer in the first and second blacks, respectively.
2. Incubate the reactions at 37 °C for 24 h and terminate the reaction by heating at 100 °C for 5 min, then centrifuge at 10,000 x g for 10 min at 4 °C.
3. Mix 300 µL of the supernatant with 600 µL DNS reagent (1:2 ratio) into new 1.5 mL microcentrifuge tubes and boil at 100 °C for 5 min.
4. Allow the solution to cool to room temperature and measure the absorbance at 540 nm using the spectrophotometer.
   NOTE: If the concentration of total reducing sugars is high in the mixture, it will result in a dark-brown solution, which is difficult to read using the spectrophotometer. Therefore, further dilution of the samples is necessary before reading the absorbance.
5. Use the glucose standard curve developed in step 6.4 above to determine the enzyme activity in the SI units of the standard curve.
6. To convert the activity to a specific activity that is expressed in µmol glucose/h/mg protein, use the equation below:
   Specific activity = [(enzyme activity/180.16)*1000]/time/protein concentration
   NOTE: Convert enzyme activity in mg/mL to g/mL; 180.16 g/mol is the molecular weight of glucose, 1000 is a factor that converts M to micromoles, time is the period of reaction, and protein extract concentration is represented in mg/mL. One unit of β-1,3-glucanase activity is defined as 1 µmol of glucose released from the substrate within a 1 h reaction period.

8. Determining Peroxidase (POD) activity

NOTE: The cell wall-bound peroxidase activity of Tugela, Tugela-Dn1, and Tugela-Dn5 wheat cultivars was determined by quantifying the formation of the tetra-guaiacol produced per unit time from guaiacol¹⁶.

1. Add 30 µL of the enzyme extract, 30 µL 1% (v/v) H₂O_2, and 970 µL of 5 mM guaiacol into the cuvettes at 25 °C.
   NOTE: Make the blank reaction without the enzyme extract (First blank), and another blank for enzyme samples without substrate (second blank). The enzyme or substrate samples were replaced by 50 mM Sodium Phosphate buffer in the first and second blanks, respectively.
2. Use the kinetic mode of the spectrophotometer to monitor the change in absorbance at 470 nm.
3. Determine the slope where the graph was linear and use it as the absorbance value.
4. Calculate the average absorbance and use it in calculating POD activity.
5. Calculate peroxidase activity using the molar extinction coefficient of guaiacol (26.6 mM^-1cm^-1) and express the activity in µmol tetra-guaiacol/min/mg protein.
6. Calculate POD activity using the equation used by Mafa et al.⁶:
   POD ACTIVITY = [(ΔABS × DF) ÷ Protein concentration] × 26.6 mM^-1cm^-1 × 1 cm
   NOTE: Where ΔABS = average absorbance; DF = Dilution factor; 26.6 mM^-1cm^-1 = Guaiacol extinction coefficient.

9. POD characterization

NOTE: POD characterization assays were conducted using 3 days post-infestation (dpi) enzyme extracts of Tugela, Tugela-Dn1, and Tugela-Dn5 following similar methods described in step 8, with minor changes in the reaction buffers and temperatures. The enzyme samples were always kept on ice while the experiments were being conducted. Run the reactions in quadruplicates with a parallel blank reaction.

1. For optimum pH assays, perform the following steps.
   1. Dissolve guaiacol substrate in 50 mM buffers (sodium citrate, pH 4 and 5), sodium phosphate (pH 6 and 7), and Tris-HCl (pH 8 and 9) (see Table of Materials).
   2. Run the reactions at 25 °C as described in step 8.
2. For optimum temperature assays, perform the following steps.
   1. Run the reactions at 25 °C, 30 °C, 40 °C, and 50 °C, respectively.
   2. Use guaiacol substrate dissolved in the buffer pH 5 (pH optimum) and run the reactions as described in step 8.
3. For thermostability assays, perform the following steps.
   1. Before starting the reactions, incubate the enzyme at 37 °C, 50 °C, and 70 °C for 30 min.
   2. Run the reactions according to the methods described in step 8 using 50 mM sodium citrate buffer (pH 5) and at 40 °C (temperature optimum).

10. β-1,3-glucanase characterization

NOTE: Conduct the characterization assays following the method described in step 7 using 3 dpi enzyme extracts, with some modifications in the reaction buffers and temperatures. Run the reactions in quadruplicates, with a parallel blank reaction for each substrate.

1. For optimum pH assays, proceed as follows:
   1. Dissolve MLG and β-1,3-glucan substrates in 50 mM buffers (sodium citrate, pH 4 and 5), sodium phosphate (pH 6 and 7), and Tris-HCl (pH 8 and 9).
   2. Run the reaction as described in step 7.
2. For optimum temperature assays, proceed as follows:
   1. Run the reactions at 25 °C, 30 °C, 40 °C, and 50 °C, respectively.
   2. Use MLG and β-1,3-glucan substrates dissolved in the 50 mM sodium citrate buffer (pH 5) with optimum pH (obtained in step 10.1) and run the reactions as described in step 7.
3. For thermostability assays, proceed as follows:
   1. Before starting the reactions, incubate the enzyme at 37 °C, 50 °C, and 70 °C for 30 min.
   2. Run the reactions using substrates dissolved in 50 mM sodium citrate buffer (pH 5) and incubate at 25 °C and 40 °C for 24 h.

11. Assessing β-1,3-glucanase mechanism of action on different glucan substrates

NOTE: Glucan substrates contain the same glucose residue in their backbone, but the glycosidic linkages between glucopyranose units are diverse and can take the α or β orientation¹⁷. The glycosidic bonds can form between several carbon atoms of glucose molecules, defining their chemical structure, e.g., β-1,3-glucan, β-1,4-glucan, and mixed linked-β-1,3-1,4-glucan (MLG)^18,19,20. The β-1,3-glucanase mechanism of action was determined using RWASA2-infestated Tugela, Tugela-Dn1, and Tugela-Dn5 samples (enzyme sources) and the following substrates β-1,3-glucan (CM-curdlan), MLG (from barley), and β-1,4-glucan (AZO-CM-Cellulose). The mechanism of action is determined under optimal assay conditions (25 °C and 40°C, pH 5.0), using 0.1% (w/v) β-1,4-glucan, 0.4% (w/v) β-1,3-glucan, or 0.5% (w/v) MLG substrates.

1. Dissolve the substrates in 50 mM sodium citrate buffer (pH 5).
2. Add 200 µL of the enzyme extract and 300 µL of the substrate into the 1.5 mL microcentrifuge tubes.
3. Incubate the reactions at two separate temperatures, 25 °C and 40 °C for 8 h.
4. Termination of the reactions by heating at 100 °C.
5. Centrifuge the reaction mixture of β-1,3-glucan and MLG at 10,000 x g for 10 min at 4 °C and proceed as described in step 7.3.
6. For β-1,4-glucan, after terminating the reaction at 100 °C, dilute the mixture with 800 µL of absolute ethanol and centrifuge at 4 °C for 10 min at 10,000 x g.
7. Transfer the supernatant into the curvets, measure the absorbance at 590 nm with the spectrophotometer, and calculate the β-1,3-glucanase activity on β-1,4-glucan using the manufacturer's procedure (see Table of Materials) and express the activity in Units/mg protein.

12. Determining β-1,3-glucanase mode of action

NOTE: The RWASA2-infested Tugela, Tugela-Dn1, and Tugela-Dn5 induced β-1,3-glucanase mode of action were assayed with laminarin-oligosaccharides (LAMs) with the degree of polymerization (DP) between 5 and 2. Use Thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), and glucose oxidase peroxidase (GOPOD) kit to determine the DP required for β-1,3-glucanase to hydrolyze the substrate. The TLC was used for qualitative analysis, and the LC-MS was used for quantitative analysis, which determined the concentration of the oligosaccharides in the hydrolysate after the reaction²¹.

1. Prepare the reactions of β-1,3-glucanase mode of action
   NOTE: Prepare the concentrated protein extract with a 10 kDa centrifuge concentrating membrane filter, and thus have concentrated and non-concentrated RWASA2 infested protein extracts for the experiment.
   1. For concentrating the enzymes extracted from each cultivar, transfer 5 mL of the extracts into the 10 kDa centrifuge concentrating membrane filters.
   2. Centrifuge the membrane filter tubes containing extract at 15,000 x g for 30 min at 4 °C.
   3. Dissolve Laminaripentaose (LAM5), Laminaritetraose (LAM4), Laminaritriose (LAM3), and Laminaribiose (LAM2) (see Table of Materials) in 50 mM sodium citrate (pH 5) to make 10 mg/mL.
   4. To start the reaction, add 20 µL of the protein extract and 40 µL of Laminaripentaose (LAM5), Laminaritetraose (LAM4), Laminaritriose (LAM3), and Laminaribiose (LAM2).
   5. Incubate the reaction at 40 °C for 16 h and terminate by boiling at 100 °C for 5 min.
2. Perform thin layer chromatography (TLC)
   1. Cut 4 silica plates into 10 cm² pieces and draw one line across the surface of the plate, leaving 1 cm space from the bottom of one edge.
   2. Mark the dots over the line and mark them according to different cultivars and DP of the Laminarin-oligosaccharides, 1 cm apart and. Take 2 silica plates for concentrated protein extract and the other 2 for non-concentrated extracts.
   3. Add 3 µL of the reaction mixtures 3 to 5 times over the corresponding marked spots on the silica plate and allow the spots on the plate to dry completely before repeating the process.
   4. Gently put the silica plates in the TLC tank containing the mobile phase of n-butanol: acetic acid: water (2:1:1 v/v/v) with the side containing the samples' spots at the bottom.
   5. Allow the mobile phase to move from the bottom to the top of the plate.
      NOTE: This step may take approximately 2 h. Do not move or disturb the tank while samples are separating.
   6. Remove the plates from the mobile phase and allow them to dry at room temperature.
   7. Add the staining solution into the small container, gently submerge the silica plate in the staining solution of 0.3% (w/v) Naphthol in 95% (v/v) ethanol using 5% (v/v) sulfuric acid for 5 s, remove it, and then allow it to dry at room temperature.
      NOTE: At this step, switch on the heating block/oven and set the temperature to 100 °C.
   8. Put the dried silica plates on the heating block and heat at 100 °C for 7-10 min or until the blue-violet spots appear. Take photos of the silica plates showing the blue-violet spots and document them.
3. Quantify the concentration of laminarin-oligosaccharides hydrolysates
   NOTE: The concentration of the LAM hydrolysates will be quantified for both concentrated and non-concentrated protein extracts²¹.
   1. Add 20 µL of each sample on a C18 (Carbohydrate 4.6 × 250 mm) column and allow to separate at a flow rate of 500 µL/min using a water (solvent A) and acetonitrile (solvent B) gradient from 100% B to 60% B over 10 min followed by column re-equilibration steps with a total run time of 20 min to allow for column re-equilibration.
   2. Ionise the eluting analytes in negative electrospray mode in the mass spectrometry ion source with a 400 °C heater temperature to evaporate the excess solvent, 30 psi nebulizer gas, 30 psi heater gas, and 20 psi curtain gas.
   3. Set the declustering potential at 350 V.
   4. Analyse the eluting analytes on the mass spectrometer in a Q1 scan mode ranging from 150 Da to 1000 Da with a dwell time of 3 s.
   5. Record the concentrations of the analytes on the spreadsheet.
4. Quantify the concentration of glucose
   1. Analyze the glucose concentration produced by laminarin hydrolysis using GOPOD reagent following the manufacturer's instructions^22,23.
   2. Measure the absorbance at 510 nm in a fixed mode of the spectrophotometer.

13. Data collection and analysis

1. Randomize all experiments to avoid bias during infestation or sample collection and conduct the analysis in quadruplicates.
2. Unless stated otherwise, use the means ± standard deviation to represent the values in the graphs and tables generated from the collected experimental data.
3. Use Microsoft Excel to analyze all the data and to generate graphs.
4. Perform statistical analysis with compatible software.
   NOTE: Perform Multifactorial Analysis of variance (ANOVA) to test for significance between treatments and Fisher's LSD to test for homogenous groups at an alpha value of 0.05.