Functional characterization of cell wall-associated β-glucanases and peroxidase induced during wheat-Diuraphis noxia inteactions.

Functional characterization of cell wall-associated β-glucanases and peroxidase induced during wheat-Diuraphis noxia inteactions. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional characterization of cell wall-associated β-glucanases and peroxidase induced during wheat-Diuraphis noxia inteactions. Siphephelo N.N. Zondo, Lintle Mohase, Vicki Tolmay, Mpho S. Mafa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3968206/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jul, 2024 Read the published version in Biologia → Version 1 posted 5 You are reading this latest preprint version Abstract Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defence responses, which include increased activity of β-1,3-glucanase and peroxidase (POD). There is a lack of information regarding β-1,3-glucanase and POD synergistic effects on the plant cell wall modification and characterisation during wheat-RWA infestation. This study aimed to characterise the physicochemical properties of the cell wall-bound POD and β-1,3-glucanase during RWA-wheat interaction. The susceptible Tugela, moderately resistant Tugela Dn1 , and resistant Tugela Dn5 cultivars were planted in a glasshouse to a seedling stage before being infested with RWASA2 for 14 days. The findings showed a significant increase in β-1,3-glucanase and POD activities in the infested Tugela Dn5 and Tugela- Dn1 cultivars over the 14 days. However, in the Tugela enzymes were repressed. In addition, it was shown for the first time that β-1,3-1,4-glucanase activity specific toward mixed-linked glucan was significant in the resistant cultivar over 14 days. β-1,3-glucanase, β-1,3-1,4-glucanase and POD displayed optimum at pH 5. β-1,3-glucanase and POD displayed temperature optimum at 40 and 50°C, respectively. However, β-1,3-1,4-glucanase had temperature optimum at 25°C. β-1,3-glucanase and POD had a thermo-stability at 37°C followed by about 80% relative activity at 70°C, but β-1,3-1,4-glucanase displayed thermostability at 25°C and retained more than 75% at 70°C, confirming that β-1,3-1,4-glucanase and β-1,3-glucanase induced in the resistant cultivars cell wall were two different enzymes. Mechanism of actions and oligosaccharide displayed that β-1,3-glucanase was highly active against β-1,3-glucan and required a triose and higher oligosaccharide to be active. Our findings demonstrated cell-wall bound POD and β-1,3-glucanase activities significantly increased in wheat after RWASA2 infestation, revealing they acted synergistically to reinforce the cell wall to deter RWASA2 feeding in resistant cultivars. β-1 3-glucanase Peroxidase wheat Russian wheat aphid wheat-pest interaction cell wall bound enzymes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defence responses, including the hypersensitive response (HR). These defence responses involve synthesising distinct pathogenesis-related (PR) proteins activated upon plant invasion by biotic stressors (Mohase and van der Westhuizen, 2002a ; Manghwar et al., 2018 ). The PR proteins, such as β-1,3-glucanase (EC. 3.2.1.39) and peroxidase (POD: EC 1.11.1.7) were significantly induced to higher levels in the resistant cultivars compared to the susceptible cultivars infested with RWA (Mohase and van der Westhuizen, 2002a ; Mohase and van der Westhuizen, 2002b ; Moloi and van der Westhuizen, 2005 ). The suggested physiological functions of β-1,3-glucanase and POD in the resistant cultivars included cell wall modification and reactive oxygen species (ROS) quenching at the cell wall and apoplastic regions during RWA infestation (Mohase and van der Westhuizen, 2002a ; Botha and Matsiliza, 2004 ; Moloi and van der Westhuizen, 2005 ; Mafa et al., 2022 ; Walker, 2022 ). The β-1,3-glucanases, PODs and chitinases (EC 3.2.1.14) are part of the most studied PR proteins during wheat-RWA interaction (Mohase and van der Westhuizen, 2002a ; Moloi and van der Westhuizen, 2005 ; Farvardin et al., 2020 ; Bhardwaj et al., 2021 ). Other PR proteins involved in plant defence reported by Appu et al. ( 2021 ), include chitosanases (EC 3.2.1.132), phenylalanine ammonia-lyase (EC 4.3.1.5) and polyphenol oxidase (EC 1.10.3.2). In the cases of β-1,3-glucanase and POD, most studies were performed using the apoplastic fluid extracted from wheat samples infested with RWA. However, there is a lack of information regarding the synergistic effect of the β-1,3-glucanase and POD on plant cell wall modification during wheat-RWA interaction. Thus, herein, we attempted to elucidate the physiological defensive roles of POD and β-1,3-glucanase on cell wall strengthening during wheat-RWA interaction. Based on the Carbohydrate-Active enZymes (CAZymes) Database ( http://www.cazy.org ), β-1,3-glucanases belong to the glycoside hydrolase (GH) family 17. They are classified into two types, i.e., endo -β-1,3-glucanases (EC 3.2.1.39) that cleave the β-1,3-linkages inside the β-glucan chain at random sites, while exo -β-1,3-glucanases (EC 3.2.1.58) cleave the β-1,3-linkages from the nonreducing end of the β-glucan chain (Rajninec et al., 2021 ; Qin et al., 2023 ). Both endo- and exo -acting β-1,3-glucanases degrade β-1,3-glucan efficiently, which regulates callose accumulation in the resistant cultivar. Other studies argued that by regulating callose, these enzymes produce β-1,3-glucan oligosaccharides (Forslund et al., 2000 ; Rajninec et al., 2021 ). The β-1,3-glucan oligosaccharides were also referred to as DAMPs (damage-associated molecular patterns), which induced pattern triggered immune (PTI) responses in plants (Forslund et al., 2000 ; Rajninec et al., 2021 ; Rebaque et al., 2021 ). The β-1,3-glucanase upregulation, alone or in collaboration with other proteins, contributed to plant resistance to RWA infestation (Wang et al., 2021 ). It is important to note that there are distinct types of beta-glucan polysaccharides in terrestrial plants; they include β-1,3-glucan; β-1,3 − 1,4 glucan, and β-1,4-glucan (Rebaque et al., 2021 ). However, it is not clear if the upregulated β-1,3-glucanase during RWA-wheat infestation is only active on one type of β-glucan or if it can also hydrolyse other types. In a study conducted by Manghwar et al. ( 2018 ), large contents of β-1,3-glucanase coding genes were expressed in resistant wheat and maize cultivars infected with Bipolaris sarokiniana , relative to controls. This observation suggests that β-1,3-glucanase is indeed significant for a plant to survive against biotic stress (including RWA infestation). Peroxidases participate in plant defence-related responses by strengthening plant cell walls through lignification, formation of intermolecular cross-links, and suberin formation (Moloi and van der Westhuizen, 2005 ; Mnich et al., 2020 ; Mafa et al., 2022 ). Peroxidase is an important biochemical marker in pest-plant interactions because its activity increases in resistant cultivars during RWA infestation (Xu et al., 2021 ). The increased peroxidase activity in resistant wheat after RWA infestation was important for the activated defence responses (Mafa et al., 2022 ). Peroxidase can either induce defensive responses or reduce the harmful effects of oxidative stress in the cell wall region (Mnich et al., 2020 ; Appu et al., 2021 ). The preceding sections of the introduction demonstrate that a lot of work has been done on the β-1,3-glucanase and POD found in the cytoplasmic and apoplastic regions. However, there are fewer or no studies that distinguished β-1,3-glucanase and POD associated with the cell wall to elucidate their synergistic role on the cell wall modification. Hence, the current study attempted to comprehensively characterize the physicochemical properties of the plant cell wall associated POD and β-1,3-glucanase in wheat cultivars infested with RWASA2. Materials and Methods Materials Tugela, Tugela Dn1 , and Tugela Dn5 seeds were supplied by the Agricultural Research Council–Small Grain (ARC-SG) Institute, Bethlehem, South Africa. A fresh colony of RWASA2 was developed on the susceptible PAN 3118 wheat cultivar in the glasshouse at UFS, Bloemfontein Campus (29°6’31.94’’ S; 26°11’18.95’’ E). The commercial barley endo -β-1,3-glucanase enzyme was purchased from Megazyme (Wicklow, Ireland). The Curdlan (β-1,3-glucan), barley mixed linked β-1,3 − 1,4-glucan (MLG), AZO-carboxymethyl cellulose (CMC), and Laminarin oligosaccharides (Laminaripentaose, Laminaritetraose, Laminaritriose, and Laminaribiose) were purchased from Megazyme (Wicklow, Ireland). The commercial guaiacol substrate was purchased from Sigma (Johannesburg, South Africa). All the chemicals for buffer preparations and all analytical chemicals used in the study were purchased from Sigma (Johannesburg, South Africa), unless stated otherwise. Plant growth conditions The trial was laid as a split-plot design, with infestation as the main plot and cultivars sub-plots and replicated four times. Plants were cultivated according to Mafa et al. ( 2022 ). Briefly, 250 seeds of susceptible Tugela, moderately resistant Tugela Dn1 and resistant Tugela Dn5 cultivars were germinated in separate Petri dishes, respectively. Germination conditions were as follows: 5 mL of distilled water was added to each Petri dish containing seeds. Petri dishes were sealed with a parafilm and incubated in a dark germination chamber at 25°C for 2 days. Tugela seeds differed from other seeds and took 7 days to germinate. The germinated seeds of each cultivar were transplanted in 15 cm pots containing 1:1 soil and peat moss (Mikskaar Professional Substrate) under controlled glasshouse conditions at the UFS, Bloemfontein Campus, South Africa. The plants were grown under a natural photoperiod and temperature regimes were 18 ± 2°C and 24 ± 2°C during night and day, respectively. The plants were irrigated every three days using filtered tap water and fertilised with 2 g/L Multifeed classic™ [5.2.4 (43)] 14 days after germination. The plants were enclosed in cages covered with nets and allowed to grow up to the second leaf stage. Russian wheat aphid (RWASA2) infestation Tugela, Tugela Dn1 , and Tugela Dn5 wheat cultivars were infested with RWASA2 according to Jimoh et al . (2013) and Mohase and Taiwe ( 2015 ). Twenty freshly harvested adult RWASA2 clone aphids were used to infest each plant at the three-leaf growth stage. The uninfested controls and infested test plants were kept in separate rooms. All plants were enclosed in cages covered with nylon nets (315 µm). There were two harvesting regimes: short-term feeding (1, 2, and 3 days) and long-term feeding (7 and 14 days). At each harvesting time ten randomly selected leaves (second and third) of each cultivar were cut at the base, wrapped in moist paper towel, immediately placed on ice and transferred to the laboratory for intercellular wash fluid (IWF) extraction. Extraction of cell wall associated β-1,3-glucanase and Peroxidase (POD) The harvested leaves were cut into 7 cm pieces. To remove any cytosolic contamination from the cut ends, the leaf pieces were rinsed twice in distilled water (Van der Westhuizen et al., 1998a ) and submerged into a 50 mM Tris-HCl extraction buffer (pH 7.8) in a thick-walled glass tube. The leaf pieces were impregnated with extraction buffer following a 5-minute infiltration under vacuum created by a water jet pump. Thereafter, leaves were removed from the glass tube, dried with a paper towel, placed vertically into pre-cooled centrifuge tubes fitted with a perforated disk, and centrifuged at 500 × g for 10 min at 4°C (Mafa et al., 2022 ). The extraction procedure was repeated, and the collected supernatant was divided into aliquots and stored at − 20°C. However, the IWF was not used in the current study, rather was extracted to ensure the enzymes studied were not apoplastic enzymes. The leaf tissue left after IWF extraction was used to extract the total protein, which was used as a source of cell wall-bound β-1,3-glucanase and POD. Leaf residues were crushed to a fine powder in liquid nitrogen using a mortar and pestle. About 200 mg of the powder was extracted with 4 mL extraction buffer (50 mM sodium phosphate, pH 6.0, containing 1% ( w/v ) polyvinylpolypyrrolidone (PVPP). The suspension was allowed to rest for 3 min and then centrifuged at 10 000 × g for 15 min. The supernatant (total proteins extracts) was collected into 2 mL Eppendorf tubes and used as source of the β-1,3-glucanase and POD. Protein concentration quantification The protein concentration of total protein extracts was determined according to Bradford ( 1976 ). Briefly, 10 µL of the enzyme extract was mixed with 190 µL Bradford reagent (BioRad) in a microplate well and incubated at 25°C for 20 min. The protein concentration was determined by measuring the absorbance at 595 nm using a microplate reader (Anthos, Zenyth 3100). Bovine serum albumin (BSA) was used as a suitable standard to develop a calibration curve with concentrations ranging between 0.05 and 1 mg/mL. β-1,3-glucanase activity assay The enzyme activity of the cell wall-bound β-1,3-glucanase extracted from the leaves was determined by measuring the amount of reducing sugars released from the 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 of Miller ( 1959 ) and Mafa et al. ( 2020 ). The reaction mixture contained 200 µL enzyme extract (protein concentrations were kept between 0.02 to 0.09 mg/mL in all reactions, unless stated otherwise), and 300 µL of the substrate dissolved in 50 mM sodium citrate buffer (pH 5). Each reaction mixture was incubated at 37°C for 24 h and the reaction was terminated by heating at 100°C for 5 min followed by centrifugation at 10 000 × g for 10 min. About 300 µL supernatant containing the reaction hydrolysate was mixed with 600 µL 3,5-Dinitrosalicylic acid (DNS) reagent at a 1:2 ratio; boiled at 100°C for 5 min and allowed to cool at room temperature. The absorbance of the samples was measured at 540 nm using a spectrophotometer (GENESYS 120 UV–Visible Spectrophotometer). Glucose was used as a standard to establish a standard curve used in determining the enzyme activity, expressed as µmol glucose/h/mg protein. One unit of β-1,3-glucanase activity was defined as the amount of enzyme that released 1 µmol of glucose/h from the substrate under the described conditions. POD activity assay A modified method of Zieslin and Ben-Zaken ( 1991 ) was used to determine cell wall-bound POD activity in the wheat cultivars. The assay mixture consisted of 970 µL of 5 mM guaiacol, 30 µL of 1% ( v/v ) H 2 O 2, and 30 µL enzyme extract. The reaction was performed at 25°C, and the change in absorbance was monitored at 470 nm using the kinetic mode of a spectrophotometer (GENESYS 120 UV–Visible Spectrophotometer). Peroxidase activity was determined using the molar extinction coefficient of guaiacol (26.6 mM − 1 cm − 1 ) and expressed in µmol tetra-guaiacol/minute/mg protein. Biochemical characterization Biochemical assays for cell wall-bound β-1,3-glucanase and POD induced in the wheat cultivars were performed to determine the pH and temperature optima, and thermostability. Based on the data analysis, the 3 dpi samples were selected to minimize bias between different cultivars’ responses to RWA infestation. POD characterization Each reaction consisted of 30 µL enzyme extract, 1% ( v/v ) H 2 O 2 and 5 mM guaiacol. The formation of the products was measured at 470 nm using the kinetic mode of the of the spectrophotometer (GENESYS 120 UV–Visible Spectrophotometer). The optimum pH studies for POD activity were conducted at 25°C using guaiacol dissolved in different 50 mM buffers, i.e., sodium citrate buffer with a pH of 4 and 5; sodium phosphate buffer with a pH of 6 and 7; and Tris-HCL buffer with a pH of 8 and 9. For optimum temperature tests, assays were conducted at 25, 30, 40 and 50°C, respectively. The reactions were conducted for 5 min using guaiacol dissolved in 50 mM sodium citrate buffer (optimum pH). For thermostability studies, the reactions were conducted by incubating the enzyme at 37, 50 and 70°C for 30 min, and reactions were conducted as described for temperature and pH optima assays. All the reactions were conducted in quadruplicates. Activity was expressed as POD relative activity using percentages and specific activity (U/mg protein). β-1,3-glucanase characterisation The MLG and β-1,3-glucan substrates were hydrolysed by the β-1,3-glucanase and the production of total reducing sugars was measured by DNS reagents as elaborated in section 3.3.7 above. The optimum pH for the enzymes was determined by adding 200 µL of the enzyme extract to 300 µL substrate dissolved in different buffers (50 mM sodium citrate pH 4 & 5; sodium phosphate pH 6 & 7; Tris-HCL pH 8 & 9) and incubating the reaction at 37°C for 24 h. For determining optimum temperature, the reactions were incubated at 25, 30, 40, and 50°C. For thermostability assays, the stability of the extracted enzyme was determined by first incubating for 30 min at 25, 37, 50, and 70°C followed by the reaction conducted as described above. All the reactions were conducted in triplicates. Activity was expressed in relative activity using percentages and specific activity (U/mg protein). β-1,3-glucanase mechanism of action assays Substrate specificity was assessed to determine further if the β-1,3-glucanase induced by RWASA2 infestation in the wheat cultivars hydrolysed β-1,3-glucan (CM-curdlan), MLG (from barley) and β-1,4-glucan (AZO-CM-Cellulose) substrates. The mechanism of action was determined under optimal assay conditions (25 & 40°C, pH 5.0) using 200 µL enzyme extract, 300 µL of the substrate [0.1% ( w/v ) β-1,4-glucan, 0.4% ( w/v ) β-1,3-glucan, or 0.5% ( w/v ) MLG] dissolved in 50 mM sodium citrate buffer (pH 5). Reactions were incubated at 25 and 40°C for 8 h, followed by termination by heating at 100°C. Proceeding reaction steps for β-1,3-glucan and MLG were conducted as described in section 3.3.7 above. For β-1,4-glucan, after terminating the reaction at 100°C, the mixture was diluted with 800 µL absolute ethanol followed by centrifugation at 10 000 × g for 10 min. After centrifugation, the absorbance was measured at 590 nm. The β-1,3-glucanase activity on β-1,4-glucan was calculated using the manufacturer’s (Megazyme) procedure and expressed in Units/mg protein. Determining β-1,3-glucanase mode of action The β-1,3-glucanase mode of action was assayed with laminarin-oligosaccharides (LAMs) with degree of polymerization (DP) between 5 and 2. Thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), and glucose oxidase peroxidase (GOPOD) were used to determine the DP required for β-1,3-glucanase to hydrolyse the substrate, to quantify the concentration of the oligosaccharides with DP between 5 and 2 and glucose, respectively (Mafa et al., 2020 ; Mafa et al., 2023 ). The total protein extract was concentrated with 10 kDa centrifuge concentrating membrane filters, and it was used as a concentrated β-1,3-glucanase sourced from samples at 3 dpi. Enzyme extracts from RWASA2-infested wheat (20 µL of concentrated or non-concentrated enzyme) were incubated at 40°C for 16 h with 10 mg/mL of Laminaripentaose (LAM5), Laminaritetraose (LAM4), Laminaritriose (LAM3), and Laminaribiose (LAM2) (Megazyme), dissolved in 50 mM sodium citrate (pH5). The reaction was terminated by boiling for 5 min before spotting the samples on the TLC. The soluble oligosaccharides (15 µL) reaction mixtures were spotted on the silica plate [TLC Silica gel 60 F 254, HPTLC plate (Merck, Darmstadt, Germany)]. TLC was conducted using the mobile phase of n-butanol: acetic acid: water (2:1:1 v/v/v ). The plate was allowed to develop for 2 h. The plate was removed from the mobile phase, dried, and stained with 0.3% ( w/v ) Naphthol (Merck, Germany) in 95% ( v/v ) ethanol using 5% ( v/v ) sulfuric acid (Merck, Darmstadt, Germany) followed by heating at 100°C for 7 min. The visible blue-violet spots were documented. The concentration of the hydrolysate (oligosaccharides) produced during hydrolysis of LAMs was determined with the LC-MS as described by Mafa et al. ( 2023 ). Hydrolysates were analysed using an ABSCIEX 4000 QTRAP hybrid triple quadrupole ion trap mass spectrometer with a Shimadzu high-performance liquid chromatography (HPLC) stack as a front end. Analyst 1.5 (AB SCIEX) software was used to perform all acquisition and processing of the data. Briefly, twenty microliters of each sample were separated on a C18 (Carbohydrate 4.6 × 250 mm, Agilent) column at 500 µL/min flow rate over 10 min using a water (solvent A) and acetonitrile (solvent B) gradient from 100% B to 60% B, followed by column re-equilibration steps with a total run time of 20 min. Eluting analytes were ionised in negative electrospray mode in the TurboV ion source with a 400°C-heater temperature to evaporate the excess solvent, 30 psi nebuliser gas, 30 psi heater gas and 20 psi curtain gas. Declustering potential was set at 350 V. Mass spectrometer was used to analyse the eluting analytes in a Q1 scan mode ranging from 150 Da to 1000 Da with a dwell time of 3 s. The glucose concentration produced by laminarin hydrolysis was further analysed using GOPOD reagent following Megazyme instructions. The absorbance was measured at 510 nm in a fixed mode of the spectrophotometer (GENESYS 120 UV–Visible Spectrophotometer). Data collection and analysis All experiments were randomised to avoid bias during infestation or sample collection, and analysis was conducted in quadruplicates. The values in the graphs and tables generated from the collected experimental data represent the means ± standard deviation unless stated otherwise. Microsoft Excel was used to analyse all the data and to generate graphs. Statistical analysis was performed with TIBCO Statistica (version 13.1), whereby multifactorial Analysis of variance (ANOVA) was performed to test for significance between treatments and the Fisher's LSD was used to test for homogenous groups at alpha value of 0.05. Results Peroxidase activity determination Peroxidase activity of the control relative to RWASA2-infested resistant Tugela Dn 5 and moderately resistant Tugela Dn 1 was not significantly different at 1 dpi, as shown in Fig. 1 . It is only in the susceptible Tugela where the POD activity in the infested was higher relative to the control. At 2, 3 and 7 dpi, RWASA2 infestation induced higher POD activity in all three cultivars than in the control treatments. In the moderately resistant Tugela Dn 1 and resistant Tugela Dn 5, RWASA2 infestation induced significantly higher POD activity relative to the control treatment at 14 dpi, showing a delayed response in the resistant cultivar. However, the opposite was observed in the susceptible Tugela cultivar (Fig. 1 A), where POD activity was repressed by RWASA2 infestation relative to the control. β-1,3-glucanase activity RWASA2 infestation significantly induced higher β-1,3-glucanase activity in the resistant Tugela Dn5 and moderately resistant Tugela Dn1. Lower activity levels were induced in the susceptible Tugela. Both RWASA2 infested and control cultivars showed that high β-1,3-glucanase activity induced in the cell wall of all three cultivars significantly increased over the 14 days post infestation (dpi) during MLG hydrolysis (Figs. 2 ). The activity of the β-1,3-glucanase extracted from Tugela did not show any specific pattern during the hydrolysis of MLG substrate and was significantly reduced at 1 and 14 dpi (Fig. 2 A). The activity of the enzyme extracted from Tugela Dn1 significantly increased at all time points, and was gradually increasing over time, except at 2 dpi. However, β-1,3-glucanase activity was significantly reduced relative to control at 1 and 2 dpi in Tugela Dn5 during the MLG hydrolysis. The activity of this enzyme seemed to be delayed showing significant increase in the samples infested for 7 and 14 days (Fig. 2 C). When the curdlan (β-1,3-glucan) was hydrolysed by β-1,3-glucanase extracted from susceptible Tugela, there were no significant differences between control and infested samples at 2 and 7 dpi, followed by significant increase activity at 3 dpi (Fig. 2 D). However, it is important to note that at 1 and 14 dpi the β-1,3-glucanase was significantly reduced compared to controls in the susceptible cultivar. The RWASA2 infested Tugela Dn 1, and control samples had comparable β-1,3-glucanase activity levels, but at 3 and 7 dpi the infested cultivar samples had significantly higher β-1,3-glucanase activity (Fig. 2 E). In contrast, this enzyme displayed significantly increased activity in the Tugela Dn5 samples from 3 to 14 dpi compared to control samples (Fig. 2 F). Overall, the results show that there are two distinct enzymes, which catalyse the MLG and β-1,3-glucan substrates such as curdlan independently. It was interesting to observe that both enzymes were significantly induced in the moderate and resistant Tugela cultivars at 3 to 14 dpi, while both enzymes’ activities were repressed in the susceptible Tugela cultivar after 3 dpi. Biochemical characterization POD characterization The biochemical conditions for POD in the susceptible Tugela, moderately resistant Tugela Dn1 , and resistant Tugela Dn5 were assessed using guaiacol substrate in the presence of H 2 O 2 , and the results displayed in Fig. 3 exhibited higher activities in acidic conditions. RWASA2 infestation induced significantly higher POD activity in Tugela Dn1 and Tugela Dn5 compared to control samples and higher activity was recorded at pH 6 and pH 5 (Fig. 3 B and C). The POD activity in the control samples was slightly higher than in infested Tugela samples (Fig. 3 A). The POD activity was significantly reduced from neutral to alkaline conditions. For instance, all samples lost more than 40 and 50% relative activity at pH 7 and 8, respectively. At pH 9, this enzyme lost over 90% relative activity in infested or controls of the three cultivars. The specific activity shows that POD activity levels were lower in Tugela (< 0.8 U/mg protein), while the enzyme activity levels were more than 1.2 U/mg protein in Tugela Dn1 and Tugela Dn5 samples. The significantly higher POD activity in moderately resistant Tugela Dn1 and resistant Tugela Dn5 samples post infestation at acidic pH range demonstrate that this enzyme was associated with the cell wall reinforcements. The optimum temperature of the POD enzyme was assessed in both the RWASA2-infested and control cultivars. Relative activity of this enzyme displayed an increasing trend from 25 to 50°C (Fig. 3 ), but the controls of Tugela and Tugela Dn5 did not show any different activity between 25 and 30°C. Infested Tugela Dn5 sample displayed significantly increased POD activity from 25 to 50°C, displaying 100% relative activity at 50°C. Similarly, RWASA2 infestation in Tugela Dn1 induced higher activity compared to control at all tested temperatures, with the maximum relative activity at 50°C. Tugela also had maximum activity for both treatments at 50°C, but the infestation reduced the POD activity by about 5% compared to the control. Once again, POD activity was significantly increased post RWA infestation in the Tugela Dn1 and Tugela Dn5 samples, while POD extracted from Tugela control and infested sample did not show any differences (Fig. 3 D, E and F). The thermostability assay was assessed by incubating the enzyme extracts at 37, 50, and 70°C for 30 min. Figure 4 showed that POD extracted from RWASA2 infested Tugela Dn1 samples retained maximum (100%) relative activity at 37°C and gradually lost activity as the temperature increased. The enzyme retained over 80% activity at 50°C. In addition, RWASA2 infested Tugela and Tugela Dn5 samples significantly reduced the POD activity compared to the control, suggesting that POD extracted from this samples was not thermo-tolerant compared to the control (Fig. 4 A and C). At 70°C, POD relative activity was lost in infested or control samples of the three cultivars, indicating that POD is a mesophilic enzyme that cannot tolerate temperatures up to 70°C. β-1,3-glucanase characterization The optimum conditions of β-1,3-glucanase activity from Tugela, Tugela Dn1 , and Tugela Dn5 were assessed using MLG and β-1,3-glucan (Curdlan) substrates (Fig. 5 ). RWASA2 infestation induced more enzyme activity relative to control in all three cultivars under acidic pH conditions. The β-1,3-glucanase showed higher enzyme activity in acidic conditions, with pH 5 showing maximum enzyme activity in all three cultivars and over 90% relative activity induced at pH 4 when MLG was used as a substrate (Fig. 5 A, B and C). At neutral pH ranges this enzyme showed a significantly decreased relative activity (retaining between 50% and 40% activity) in Tugela and Tugela Dn1 , while it lost more than 60% relative activity in Tugela Dn5 samples from pH 6 to pH 9. In addition, the specific activity showed that Tugela Dn5 has higher levels of β-1,3-glucanase activity (reaching more than 20 U/mg protein) compared to moderately resistant or susceptible cultivars. When the β-1,3-glucanase pH optima studies were conducted using the Curdlan, the relative activity profile of all three cultivars differed from the one displayed when MLG was used as a substrate. It is also worth noting that infestation induced β-1,3-glucanase activity in Tugela Dn1 and Tugela Dn5 over acidic pH range (Fig. 5 D, E and F). In addition, the enzyme showed the optimum pH at 5 in the RWASA2 infestation samples and controls. The enzyme lost more than 60% relative activity in the Tugela Dn1 and Tugela Dn5 from pH 8 and pH 9, while Tugela samples lost more than 70% at the same alkaline pH range. The results in Fig. 5 demonstrated that the enzymes hydrolysed curdlan and MLG were two different enzymes. This observation supports the earlier specific activity result, suggesting that this enzyme could be MLG specific β-glucanase or two distinctly different β-1,3-glucanase isozymes. The optimum temperature of β-1,3-glucanase enzyme extracted from RWASA2-infested wheat cultivars was assessed using temperatures ranging from 25 to 50°C. Figure 6 results showed that during the hydrolysis of MLG substrate, the β-1,3-glucanase displayed maximum enzyme activities at 25°C in Tugela, Tugela Dn1 and Tugela Dn5 samples. The enzymes retained more than 90% relative activities in Tugela Dn1 and Tugela Dn5 samples at 40 and 50°C, but about 80% relative activity was retained in Tugela samples. Therefore, 25°C was the optimum temperature for β-1,3-glucanase on the MLG substrate. In addition, the specific activity levels of the β-1,3-glucanase in the Tugela Dn5 were significantly high compared to the control (Fig. 6 C). Also, β-1,3-glucanase specific activity levels in Tugela Dn5 was 2-fold higher than those displayed by Tugela Dn1 and 4-fold higher than those displayed by Tugela. During the hydrolysis of curdlan substrate, β-1,3-glucanase showed an optimum temperature at 40°C for the three wheat cultivars. At 50°C the enzyme retained more than 80% relative activity in the RWASA2-infested Tugela, Tugela Dn1 and Tugela Dn5 samples (Fig. 6 D, E and F). Once again, Tugela Dn1 and Tugela Dn5 β-1,3-glucanase specific activities were significantly higher in the infested samples compared to controls. However, no significant differences were observed between infested Tugela and control samples. The enzyme specific activities were similar in Tugela Dn1 and Tugela Dn5 (reaching values around 7.5 U/mg protein). At 40°C, the RWASA2 infested moderately resistant and resistant cultivars showed 3-fold higher specific activity compared to controls. The results of Fig. 6 displayed two different catalytic patterns when the enzyme acted on the MLG and curdlan, suggesting there were two different enzymes or β-1,3-glucanase isozymes or MLG specific glucanase and β-1,3-glucanase. The thermostability assay was conducted by incubating the enzyme extract at 25, 37, 50, and 70°C for 30 min. The percentage relative activity results showed that β-1,3-glucanase enzyme extracted from RWASA2-infested Tugela Dn5 and Tugela Dn1 samples was significantly thermostable compared to the control (Fig. 7 B and C) hydrolysing MLG. In addition, Tugela Dn1 and Tugela Dn5 infested with RWASA2 induced the highest β-1,3-glucanase specific activity at 25°C during the hydrolysis of MLG. Figure 7 B and C results showed that at 50 and 70°C the β-1,3-glucanase extracted from Tugela Dn1 and Tugela Dn5 retained over 80% relative activity during the hydrolysis of MLG. These results confirm that the enzymes catalysing the MLG substrates were thermostable in resistant cultivars. In contrast, the thermostability assay of β-1,3-glucanase extracted from the three wheat cultivars showed that it is a mesophilic enzyme during the hydrolysis of β-1,3-glucan substrate. At 37°C, the enzyme extracted from Tugela, Tugela Dn1 , and Tugela Dn5 infested with RWASA2 displayed over 80% relative activity. The β-1,3-glucanase activity increased as the temperature increased to 50°C in all three cultivars. Once again, RWASA2 infested Tugela Dn1 and Tugela Dn5 induced significantly higher enzymatic activities relative to controls. Also, results in Fig. 7 D, E and F showed that β-1,3-glucanase displayed significant reduction in enzyme activity at 70°C in the infested and controls samples of the three wheat cultivars. These findings show that β-1,3-glucanase enzyme that hydrolyses the curdlan (β-1,3-glucan) substrate is not a thermostable enzyme, but a mesophilic enzyme, while the MLG specific enzyme was thermostable. β-1,3-glucanase mechanism of action Mechanism of action is a study that explains the glycosidic linkages required for the enzyme to be active. The previous results sections showed that the enzymes acting on MLG and curdlan substrates differed. This observation warranted further investigation to probe if the β-1,3-glucanase has similar activity on glucan substrates with different glycosidic linkages (i.e., MLG, curdlan and CMC substrates) under optimum condition. Figure 8 A shows β-1,3-glucanase activity on β-1,3 − 1,4-glucan substrate. The enzyme from Tugela Dn5 had higher activity than the one extracted from Tugela Dn1 and Tugela, showing more activity at 25°C than at 40°C. In addition, β-1,3-glucanase extracted from Tugela showed significantly lower activity levels during hydrolysis of curdlan (β-1,3-glucan) substrate. In contrast, Tugela Dn1 displayed the highest enzyme activity at 40°C during curdlan hydrolysis (Fig. 8 B). The RWASA2-infested resistant Tugela Dn5 β-1,3-glucanase activity levels were induced 3-fold more during the hydrolysis of the substrate with β-1,3-glycosidic linkages compared to when it hydrolysed the substrate with β-1,4-glycosidic linkages; and 2-folds more activity compared to when it hydrolysed the β-1,3 − 1,4-glycosidic linkage. These findings support our claim that there are two different β-1,3-glucanase isozymes or β-1,3-glucan and MLG specific enzymes, which are significantly induced by RWASA2 in Tugela Dn5 . Hydrolytic patterns of β-1,3-glucanase Laminari-oligosaccharides which included laminaripentaose (LAM5), laminaritetraose (LAM4), laminaritriose (LAM3), and laminaribiose (LAM2) were hydrolysed with β-1,3-glucanase extracted from the RWASA2 infested Tugela, Tugela Dn1 and Tugela Dn5; the reaction was conducted for 16 h and the hydrolysate was analysed with TLC. The β-1,3-glucanase without the oligosaccharides (control) showed a single band of monosaccharide in all three cultivars, corresponding to the glucose molecule. The β-1,3-glucanase extracted from Tugela Dn5 hydrolysed the LAM5 to LAM2 efficiently, producing glucose (Fig. 9 ). LAM5 hydrolysis produced higher amounts of glucose, followed by LAM3 and LAM2. The enzyme extracted from Tugela Dn1 samples was the second most effective in hydrolysing LAM5, LAM4, LAM3, and LAM2 into glucose. It also produced a similar oligosaccharide pattern to the enzyme extracted from the resistant Tugela Dn5 cultivar. The least effective enzymes were extracted from Tugela samples, and they showed higher activity on LAM5 compared to shorter oligosaccharides (Fig. 9 ). Laminarin-oligosaccharides hydrolysates analysed with LC-MS The laminarin-oligosaccharides with the DP between 5 and 2 were hydrolysed with β-1,3-glucanase extracted from the wheat cultivars infested with RWASA2 for 3 days and the reaction products were quantified by LC-MS and GOPOD reagents. Table 3.1 showed that the non-concentrated enzymes could hydrolyse the LAM5 into glucose (green colour of the heat map), followed by a moderate concentration of LAM4 (yellow colour). The heat map shows that the LAM3 and LAM2 produced during the LAM5 hydrolysis were in trace concentration (red colour). The non-concentrated enzyme extracted from Tugela, Tugela Dn1 and Tugela Dn5 did not hydrolyse the substrate oligosaccharides efficiently, leaving them with high concentrations ranging between 183 and 122 mg/L (Table 1 green colour). In contrast, Table 2 shows the results of the concentrated β-1,3-glucanase sourced from the Tugela Dn5 , Tugela Dn1 and Tugela at 3 dpi. Tugela Dn1 β-1,3-glucanase was highly effective during the hydrolysis of Laminarin-oligosaccharides with DP between 5 and 2. This enzyme showed the highest activity on the longer oligosaccharides (LAM5 and LAM4), leaving about 9.6 and 15.9 mg/L, respectively. However, it showed moderate activity during the hydrolysis of shorter oligosaccharides (LAM3 and LAM2), leaving about 40.2 and 66.8 mg/L, respectively. The Tugela Dn1 β-1,3-glucanases produced a higher glucose concentration, followed by tetraose and biose during the hydrolysis of LAM5 to LAM2 (Table 2 ). The concentrated enzyme extracted from Tugela Dn5 and Tugela also hydrolysed the Laminarin-oligosaccharides with higher DP (LAM5 and LAM4) efficiently compared to non-concentrated enzymes. Table 1 . Laminarin-oligosaccharides produced with non-concentrated β-1,3-glucanase extracted from wheat cultivars infested with RWASA2 for 3 days. Table 2 . Laminarin-oligosaccharides produced with concentrated β-1,3-glucanase extracted from wheat cultivars infested with RWASA2 for 3 days. Discussion During RWA infestation, resistant wheat plants induce the upregulation of POD and β-1,3-glucanase activities throughout treatment to modify the cell wall by regulating callose and lignin accumulation (Forslund et al., 2000 ; Miedes et al., 2014 ; Rajninec et al., 2021 ; Mafa et al., 2022 ). Other studies suggested that the β-1,3-glucanase regulates callose by degrading it into oligosaccharides referred to as DAMPs, which induce plant innate immune response (Forslund et al., 2000 ; Luna et al., 2011 ; Rebaque et al., 2021 ). It is important to note that the majority of the studies reported in the literature used and characterised the apoplastic POD and β-1,3-glucanase (Forslund et al., 2000 ; Mohase and van der Westhuizen, 2002a , b ; Moloi and van der Westhuizen, 2005 ; Mafa et al., 2022 ). However, there is a lack of knowledge concerning the characterisation and physiological functions of cell wall-bound/associated POD and β-1,3-glucanase. Herein, the POD and β-1,3-glucanase associated with the cell wall were extracted from the cell wall region of RWASA2 infested susceptible Tugela, moderately resistant Tugela Dn1 and resistant Tugela Dn5 to study their substrate specificity and characterisation to understand their functions better. The results show that RWASA2 infestation induced significantly higher POD and β-1,3-glucanase activity levels in the cell wall regions of the moderately resistant and resistant wheat cultivars. β-1,3-glucanases were highly induced during wheat-RWA interactions (e.g. Diuraphis noxia ) or wheat-pathogen (e.g. Fusarium graminearum ) interaction, which is associated with immune responses in wheat cultivars (Mohase and van der Westhuizen, 2002a ; Van der Westhuizen et al., 2002 ; Mohase and Taiwe, 2015 ; Zhang et al., 2018 ). Several studies used IWF extraction methods to collect the apoplastic fluid containing, among other proteins, β-1,3-glucanases and POD (van der Westhuizen et al., 2002 ; Moloi and van der Westhuizen, 2005 ; Mohase and Taiwe, 2015 ). These enzymes were linked to resistant responses during RWA-wheat interactions because they were significantly induced in the resistant cultivars compared to control. For example, Van der Westhuizen et al. ( 2002 ) reported that apoplastic β-1,3-glucanase accumulated in the RWASA1-infested resistant Tugela Dn1 played a significant role in the defence mechanism. However, the authors reported that the role of β-1,3-glucanase in defence against RWA was not clear (van der Westhuizen et al., 2002 ). Other studies argued that β-1,3-glucanase regulates callose accumulation and mitigates the phloem and plasmodesmata channels clogging, allowing a more efficient cell-to-cell communication (Botha et al., 2005 ; Pirselova and Matusikova, 2012 ; Silva-Sanzana et al ., 2019; Walker, 2022 ). To the best of our knowledge, the current study is the first to extract the cell wall-bound β-1,3-glucanase from the susceptible, moderately resistant and resistant wheat cultivars infested with RWASA2. The β-1,3-glucanase extracted from the moderately resistant Tugela Dn1 and resistant Tugela Dn5 hydrolysed the MLG and curdlan (β-1,3-glucan). These observations indicated that the cell wall extracted total protein contained β-1,3-glucanase which could hydrolyse the MLG or contained MLG specific glucanase enzyme. The β-1,3-glucan specific enzyme was possibly involved in callose regulation, while MLG specific enzyme modified the hemicellulose content of the resistant wheat cultivars infested with RWASA2. Bourdon et al. ( 2023 ) used wild-type and mutants of poplar plants to demonstrate that the callose deposition in the cell wall regions led to cellulose microfiber hydration, resulting in cellulose microfibril aggregates. These microfibril aggregations created gaps (mesopores with sizes ranging from 4 to 32 nm) between the cell wall polysaccharides and gaps between polysaccharides-lignin regions. Callose is semi-permeable, and its hygroscopic properties may impact the diffusion of monolignols, laccases and peroxidases necessary for subsequent lignin polymerization. Additionally, Bourdon et al. ( 2023 ) proposed model indicated that lignin content was reduced in the cell wall regions close to callose aggregates between the microfibrils. Mafa et al. ( 2022 ) showed that the resistant wheat cultivars infested with RWASA2 induced significantly higher apoplastic POD and β-1,3-glucanase activity levels, which were correlated to the cell wall reinforcement. The current study shows that in addition to the apoplastic enzymes, there are cell wall-bound POD and β-1,3-glucanase enzymes, which showed significantly higher activity levels upon RWASA2-wheat interactions. The significantly higher POD activity levels were also reported in previous studies (Verma et al., 2017 ; Moloi and van der Westhuizen, 2005 ; Mafa et al., 2022 ). They reported a significant increase in POD activity, possibly involved in cell wall stiffening and strengthening by cross-linking the lignin content or lignin-hemicellulose connections. Ostergaard et al. ( 2000 ) proposed that POD activity in the cell wall is involved in the polymerisation of monolignols into lignin during cell wall lignification. In addition, Verma et al. ( 2017 ) observed that POD isozymes were abundant in the plant cell wall region and linked them to plant defensive responses. These observations support findings of the current study, which show the significantly higher activity of the POD extracted from the cell wall region of moderately resistant Tugela Dn1 and resistant Tugela Dn5 infested with RWASA2 for 14 days. The biochemical characterisation revealed that POD had optimum activity in the acidic pH range (pH5) for the Tugela, Tugela Dn1 and Tugela Dn5 samples. However, Tugela Dn1 and Tugela Dn5 extracted POD showed a broader pH optimum because they also showed optimum activity at pH 6. Peroxidase from Tugela Dn1 leaves displayed an optimum pH of 5.6 (van der Westhuizen et al., 1998b ). In addition, soybean and lettuce stem POD displayed optimum activity at pH 5 (Hu et al., 2012 ; Feltrin et al., 2017 ). Although the studies did not specify if they were cell wall or cytoplasmic POD, their optimum pH was comparable to the cell wall-bound POD. All samples lost more than 70% of enzyme activity in the alkaline pH range. Other studies showed that cytoplasmic POD had an optimum activity at pH 7 (neutral range) in wheat and tobacco samples (Bania and Mahanta, 2012 ; Hamid et al., 2016 ). This observation validated that the POD from the three wheat cultivars infested with RWASA2 were extracted from the cell wall region, which is generally acidic. In addition, β-1,3-glucanase extracted from RWASA2-infested Tugela, Tugela Dn1 and Tugela Dn5 displayed optimal activity at pH 5 and retained 90% relative activity at pH 4. Taiz and Jones ( 1970 ) demonstrated that β-1,3-glucanase on barley seed displayed optimal activity at an acidic pH range between 4.47 to 5.5 using laminarin as a substrate. Wheat apoplastic β-1,3-glucanase displayed optimum activity at pH 6.5 (van der Westhuizen et al., 2002 ; Zhang et al., 2018 ). Our findings showed that the β-1,3-glucanase extracted from the three wheat cultivars were cell wall related because they showed optimum activity at pH5 (and retained most activity at the acidic pH range) during curdlan and MLG substrates hydrolysis. The temperature studies displayed a gradual increase in POD activity, reaching optimum at 50°C in the three wheat cultivars. The optimum temperature profile was similar to the POD extracted from wheat (Hamid et al., 2016 ; Altin et al., 2017 ). The β-1,3-glucanase optimum temperature was investigated in both the β-1,3-glucan and β-1,3 − 1,4-glucan substrates. It displayed maximum enzyme activity at 40°C when hydrolysing curdlan and 25°C during the hydrolysis of MLG. The β-1,3-glucanase retained more than 80% relative activity at 40 and 50°C during the hydrolysis of MLG substrate. However, Zhang et al. ( 2018 ) reported β-1,3-glucanase displaying maximum activity at 50°C. In addition, the β-1,3-glucanase sourced from tomato roots displays optimum temperature at 40°C (Mohamed et al., 2007 ). These findings confirm the observation that the protein extracts contained two isozymes or β-1,3-glucanase and MLG specific enzymes. The thermostability assays for β-1,3-glucanase also validated our claim, showing that Tugela Dn1 and Tugela Dn5 extracted enzymes were thermostable (retaining more than 70% relative activity at 70°C). Future research should elucidate the function of the MLG specific β-1,3-glucanase or β-1,3 − 1,4-glucanase enzymes bound to the cell wall region. β-1,3-glucanase mechanism of action was assessed with mixed-linked glucan, curdlan and Azo-CMC substrates, representing β-1,3 − 1,4-, β-1,3- and β-1,4-glycosidic-linkages, respectively. The enzymes showed a higher activity towards a substrate that was constituted by β-1,3-glycosidic bonds compared to β-1,4-glycosidic bonds or mixed linked β-1,3 − 1,4-glycosydic bonds because it displayed higher activity on curdlan, followed by MLG and CMC substrates. Rodriguez-Mendoza et al . (2019) studied β-1,3-glucanase displaying higher affinity and activity towards β-1,3-linkages of laminarin and curdlan. β-1,3-glucanase showed higher activity on laminarin and curdlan because they are chemically similar to callose, which is made up of glucopyranosyl units joined by β-1,3-glycosidic bonds (Moravcikova et al., 2016 ; Mafa et al., 2022 ; Walker, 2022 ). To the best of our knowledge, a few studies have investigated the preference of β-1,3-glucanases activity toward substrates made of different β-1,4-glycosidic-linkage, β-1,3-glycosidic-linkage or β-1,3 − 1,4-glycosidic linkages. Because the β-1,3-glucanase from RWASA2 infested moderately resistant and resistant Tugela cultivars was significantly highly active compared to controls and susceptible cultivars, we investigated the mode of action of this enzyme using LAM5 to LAM2 as substrates. Tugela Dn1 and Tugela Dn5 extracted β-1,3-glucanase were significantly effective on oligosaccharides with longer DP according to the TLC and LC-MS results. The susceptible cultivar had the lowest activity on all laminarin-oligosaccharides. Driskill et al. ( 1999 ) corroborated our findings that the β-1,3-glucanase displayed the highest hydrolytic activity on oligosaccharides with higher DP (e.g. LAM3 to LAM6). In addition, a β-glucanase with broad glucan substrate specificity sourced from a fungus ( Podospora anserine ) showed the highest hydrolytic activity on laminarin, followed by LAM3 to LAM6, in that order (Lafond et al., 2012 ). Conclusion Previous studies have demonstrated that the RWA-wheat interaction led to upregulation of defence-related proteins in wheat plants. Among these proteins, the apoplastic and cytoplasmic POD and β-1,3-glucanase were demonstrated to play a physiological function of modulating ROS levels and regulating callose accumulation in the infested resistant wheat cultivars, respectively. Herein, we demonstrated that cell wall-bound POD extracted from RWASA2-infested moderately resistant Tugela Dn1 and resistant Tugela Dn5 cultivars had significantly higher activity compared to the control. In contrast, this enzyme displayed significantly reduced activity over time in the susceptible cultivars. This finding confirmed that the cell wall-bound POD has a defensive role against RWA infestation in the resistant wheat cultivars. In addition, two β-glucanase enzymes displayed significantly higher activity in resistant cultivars than controls during RWASA2 infestation. To our knowledge, this is the first study that links MLG specific cell wall bound β-glucanase to wheat defence responses upon RWA infestation. The characterisation confirmed that the enzymes studied were cell wall-bound because they had optimum pH at the acid range (pH). The temperature and thermo-stability assays showed that POD and β-1,3-glucanase had similar profiles, displaying optimum temperature at 40 and 50°C, respectively. Also, both enzymes were thermophilic, losing more than 90% activity at 70°C. In contrast, the MLG specific β-glucanase displayed temperature optimum at 25°C but retained more than 80% activity at 40°C in Tugela Dn1 and Tugela Dn5 , confirming that this enzyme was different from β-1,3-glucanase. In addition, the mechanism of action analysis confirmed that the MLG specific β-glucanase and β-1,3-glucanase were associated with the wheat defence responses against RWASA2 infestations because their activity levels significantly increased in the resistant Tugela Dn5 . In conclusion, the characterisation findings suggest that POD and β-1,3-glucanase function under the same conditions, suggesting they synergistically act to reinforce the cell wall during the RWASA2 infestation. Declarations Acknowledgements M. Mafa received funding from the NRF-Thuthuka (Reference Number: TTK2204102938). S.N. Zondo received the National Research Foundation Postgraduate Scholarship for his MSc degree. Authors are grateful to Agricultural Research Council – Small Grain (ARC-SG) Institute for providing the seeds used in this study. Any opinion, findings, and recommendations expressed in this material are those of the author(s), and therefore, the funders do not accept any liability in regard thereto. Authors’ contributions M. Mafa, and S.N. Zondo conceived and designed research. M. Mafa sourced funds and supervised the study. S.N. Zondo conducted experiments and wrote the wrote first draft. M. Mafa, S.N. Zondo, L. Mohase and V. Tolmay analysed and interpreted data. L. Mohase and V. Tolmay Co-supervised the study. S.N. Zondo and Mafa wrote the first draft of the manuscript. All authors read, edited and approved the manuscript. Data Availability Experimental datasets used in the current study is available from the corresponding author on reasonable request. Declarations Conflicts of interest The authors declare that they have no conflict of interest. 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Moravcikova, J., Margetinyova, D., Galova, Z., Zur, I., Gregorova, Z., Zimova, M., Boszoradova, E., and Matusikova, I., 2016. Beta-1,3-Glucanase Activities in Wheat and Relative Species. Nova Biotechnologica et Chimica . 15(2), 122–132. Ostergaard, L., Teilum, K., Mirza, O., Mattsson, O., Petersen, M., Welinder, K.G., Mundy, J., Gajhede, M., and Henriksen, A., 2000. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification. Plant Molecular Biology . 44, 231–243. Pirselova, B., and Matusikova, I., 2012. Callose: the plant cell wall polysaccharide with multiple biological functions. Acta Physiologia Plantarum . 35, 635–644. Qin, Z., Yu, S., Zhang, K., Wei, X., Li, J., Zhang, Z., Wan, S., and Gao, H., 2023. Characterization of a glycoside hydrolase family 157 endo -β-1, 3-glucanase that displays antifungal activity against phytopathogens. Journal of Agricultural and Food Chemistry , 71(27), 10383–10392. Rajninec, M., Fratrokova, M., Boszoradova, E., Jopcik, M., Bauer, M., and Libantova, J., 2021. Basic β-1,3-glucanse from drosera binate exhibits antifungal potential in transgenic tobacco plants. Plants . 10(8), 1747. Rebaque D., del Hierro I., Lopez G., Bacete L., Vilaplana F., Dallabernardina P., Pfrengle F., Jorda L., Sanchez-Vallet A., Perez R., Brunner F., Molina H., and Melida H., 2021. Cell wall-derived mixed-linked β-1,3/1,4-glucans trigger immune responses and disease resistance in plants. The Plant Journal . 106(3), 601–615. Rodríguez-Mendoza, J., Santiago-Hernández, A., Alvarez-Zúñiga, M.T., Gutiérrez-Antón, M., Aguilar-Osorio, G., and Hidalgo-Lara, M.E., 2019. Purification and biochemical characterization of a novel thermophilic exo -β-1,3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1. Electronic Journal of Biotechnology . 41, 60–71. Silva-Sanzana, C., Estevez, J.M., and Blanco-Herrera, F., 2020. Influence of cell wall polymers and their modifying enzymes during plant–aphid interactions. Journal of Experimental Botany . 71(13), 3854–3864. Taiz, L., and Jones, R.L., 1970. Gibberellic acid, β-1,3-glucanase and the cell walls of barley aleurone layers. Planta . 92, 73–84. Van der Westhuizen, A.J, Qian, X.M, and Botha, A.M., 1998a. β-1,3- glucanases in wheat and resistance to the Russian wheat aphid. Physiologia Plantarum . 103(1), 125–131. Van der Westhuizen, A. J., Qian, X. M., and Botha, A. M., 1998b. Differential induction of apoplastic peroxidase and chitinase activities in susceptible and resistant wheat cultivars by Russian wheat aphid infestation. Plant Cell Reports . 18, 132–137. Van der Westhuizen, A.J., Qian, X-M., Wilding, M., and Botha, A-M., 2002. Purification and immunocytochemical localization of wheat β-1,3-glucanase induced by Russian wheat aphid infestation. South African Journal of Science . 98, 197–202. Verma, H., Sakhre, A.S., and Singh, V.P., 2017. Assay of Peroxidase (POX) activity. Manual of ICAR Sponsored Training Programme for Technical Staff of ICAR Institutes on “Physiological Techniques to Analyze the Impact of Climate Change on Crop Plants” . 92. Walker, G.P., 2022. Sieve element occlusion: Interaction with phloem sap-feeding insects. A review. Journal of Plant Physiology . 269, 153582. Wang Y., Li X., Fan B., Zhu C., and Chen Z., 2021. Regulation and function of defense-related callose deposition in plants. International Journal of Molecular Sciences . 22(2393). Xu Y., Guo H., Geng G., Zhang Q., and Zhang S., 2021. Changes in defense-related enzymes and phenolics in resistant and susceptible common wheat cultivars under aphid stress. Acta Physiologiae Plantarum . 43(36). Zhang, S., Zhang, W., Zhai, H., Lv, Y., Cai, J., Jia, F., Wang, J., and Hu, Y., 2018. Expression of wheat β-1,3-glucanase in Pichia pastoris and its inhibitory effect on fungi commonly associated with wheat kernel. Protein Expression and Purification . 154, 134–139. Zieslin, N., and Ben-Zaken, R., 1991. Peroxidases, phenylalanine ammonia-lyase and lignification in peduncles of rose flowers. Plant physiology and biochemistry (Paris) . 29(2),147–151. Cite Share Download PDF Status: Published Journal Publication published 22 Jul, 2024 Read the published version in Biologia → Version 1 posted Editorial decision: Major revisions 11 Apr, 2024 Reviewers agreed at journal 04 Mar, 2024 Reviewers invited by journal 01 Mar, 2024 Editor assigned by journal 25 Feb, 2024 First submitted to journal 21 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3968206","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275963453,"identity":"c13e2baa-c8a1-4055-bdf2-15660de6ae5a","order_by":0,"name":"Siphephelo N.N. Zondo","email":"","orcid":"","institution":"University of the Free State - Bloemfontein Campus: University of the Free State","correspondingAuthor":false,"prefix":"","firstName":"Siphephelo","middleName":"N.N.","lastName":"Zondo","suffix":""},{"id":275963454,"identity":"6501b780-87fd-47a5-9229-c1a0ba4be903","order_by":1,"name":"Lintle Mohase","email":"","orcid":"","institution":"University of the Free State - Bloemfontein Campus: University of the Free State","correspondingAuthor":false,"prefix":"","firstName":"Lintle","middleName":"","lastName":"Mohase","suffix":""},{"id":275963455,"identity":"7a751096-bc73-4e70-9ec5-d713358a9049","order_by":2,"name":"Vicki Tolmay","email":"","orcid":"","institution":"Agricultural Research Council","correspondingAuthor":false,"prefix":"","firstName":"Vicki","middleName":"","lastName":"Tolmay","suffix":""},{"id":275963456,"identity":"32365ac7-14f6-4be1-a529-4cccaf827775","order_by":3,"name":"Mpho S. Mafa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYFACxgdAwoaBvYF4LcwGQCKNgecAiVoOk6BFt/0w46cbf84n9vAfYPzwg6FOnqAWszPJzNK5bbcTeyQSmCV7GNgMGwhqOZB/QDq34XbifgkGBmkGBh5GwlrOP2b+nfPnHMhhzL8ZGCTsCWu5kcwmncN2ILGHIYENaItBIhFaHrNZ57YlG/dIJLZZ9hgkJBPhsGTm2zl/7GR7+A8fvvGjos6WoBYkAPK4AQnqR8EoGAWjYBTgBgDlPznk+YdXDwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2385-4423","institution":"University of the Free State","correspondingAuthor":true,"prefix":"","firstName":"Mpho","middleName":"S.","lastName":"Mafa","suffix":""}],"badges":[],"createdAt":"2024-02-18 22:48:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3968206/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3968206/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11756-024-01734-1","type":"published","date":"2024-07-22T16:16:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52083222,"identity":"716aa21a-1e72-4976-b84b-0899bd3dae93","added_by":"auto","created_at":"2024-03-06 11:32:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44230,"visible":true,"origin":"","legend":"\u003cp\u003eThe specific activity of peroxidase extracted from RWASA2 infested Tugela (A), Tugela \u003cem\u003eDn1\u003c/em\u003e (B) and Tugela \u003cem\u003eDn5\u003c/em\u003e(C) 14 days after infestation. The experiments were performed in quadruplicates; the values and error bars represent means ±SD, respectively. U in U/mg protein represents μmol tetra-guaiacol/min.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/15769eda7db6cc38fff2e9ca.png"},{"id":52083221,"identity":"b43e3cad-0ba4-4fb9-adc3-ced5323b953d","added_by":"auto","created_at":"2024-03-06 11:32:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76602,"visible":true,"origin":"","legend":"\u003cp\u003eThe β-1,3-glucanase specific activity measured in RWASA2 infested Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e cultivars. The enzyme activity was conducted using two chemically distinct glucan substrates, i.e., mixed-linked β-1,3-1,4-glucan (A, B, C) and curdlan (D, E, F). The experiments were performed in quadruplicates; the values and error bars represent means ±SD, respectively. U represents μmol/h.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/f675ab0905b8ae5b3d4ed1cf.png"},{"id":52083538,"identity":"2f4aaa18-7f36-47e4-9de9-c799d7e39786","added_by":"auto","created_at":"2024-03-06 11:40:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116321,"visible":true,"origin":"","legend":"\u003cp\u003eThe optimum pH (A, B, C) and temperature (D, E, F) assays of the peroxidase extracted 3 days after RWASA2 infestation in Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e. The experiments were performed in quadruplicates; the values and error bars represent means ±SD, respectively. The line graph represents the percentage relative activity, and bar graph represents specific activity. U represents μmol tetra-guaiacol/min.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/5996563e23acaf97b50eb20d.png"},{"id":52083224,"identity":"96273739-6409-42ae-a741-2398e3c8972b","added_by":"auto","created_at":"2024-03-06 11:32:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70275,"visible":true,"origin":"","legend":"\u003cp\u003eThermostability assays of peroxidase enzyme extracted from RWASA2 infested Tugela (A), Tugela \u003cem\u003eDn1\u003c/em\u003e (B) and Tugela \u003cem\u003eDn5\u003c/em\u003e (C) at 3 days post infestation. The experiments were performed in quadruplicates; the values and error bars represent means ±SD, respectively. The line graph represents relative activity, and the bar graph represents specific activity. U represents μmol tetra-guaiacol/min.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/31f09cf910e4ddfc321619d1.png"},{"id":52083978,"identity":"36aa6a0e-0b9e-44fe-9f5b-0335d8f00fb8","added_by":"auto","created_at":"2024-03-06 11:48:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":121059,"visible":true,"origin":"","legend":"\u003cp\u003eThe pH optimum assays of β-1,3-glucanase enzyme extracted from RWASA2 infested Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003eat 3 days after infestation. The experiments were conducted using mixed-linkage-β-1,3-1,4-glucan (A, B, C) and curdlan (D, E, F) substrates. The experiments were performed in quadruplicates; the values and error bars represented means ± SD, respectively. The line graph represents relative activity, and bar graph represents specific activity. U represents μmol/h.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/d855415ccf4df89f44e08891.png"},{"id":52083227,"identity":"08f665d2-1300-48a4-9166-ecc59a443545","added_by":"auto","created_at":"2024-03-06 11:32:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":125257,"visible":true,"origin":"","legend":"\u003cp\u003eThe temperature optimum assays of β-1,3-glucanase enzyme extracted from RWASA2 infested Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003eat 3 days after infestation. The experiments were conducted using mixed-linkage-β-1,3-1,4-glucan (A, B, C) and curdlan (D, E, F) substrates. The experiments were performed in quadruplicates; the values and error bars represented means ±SD, respectively. The line graph represents relative activity, and the bar graph represents specific activity. U represents μmol/h.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/3dba5a7254e0dea91f876256.png"},{"id":52083225,"identity":"35fbf786-5ba4-4903-9d6f-99ce61397764","added_by":"auto","created_at":"2024-03-06 11:32:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":130447,"visible":true,"origin":"","legend":"\u003cp\u003eThe thermostability assays of β-1,3-glucanase sourced from RWASA2 infested Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e 3 days after infestation, investigated on β-1,3-1,4-glucan (A, B, C) and β-1,3-glucan (D, E, F). The experiments were performed in quadruplicates; the values and error bars represent means ± SD, respectively. The line graph represents relative activity, and the bar graph represents specific activity. U represents μmol/h.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/1d7902d2e4869d935ec89c85.png"},{"id":52083223,"identity":"0f28239d-bbab-4dc1-862d-b1e0543c41eb","added_by":"auto","created_at":"2024-03-06 11:32:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":45414,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the mechanism of action for β-1,3-glucanase extracted from RWASA2 infested Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e. Experiments were conducted using glucan substrates with different chemical glycosidic linkages, i.e., Mixed linkage- β-1,3-1,4-glucan (A), curdlan\u003cstrong\u003e \u003c/strong\u003e(β-1,3-glucan) (B) and\u003cstrong\u003e \u003c/strong\u003eCMC (β-1,4-glucan) (C) substrates. The assays were conducted at both 25 and 40°C, and optimum pH 5. All experiments were performed in quadruplicates; the values and error bars represent the mean ±SD, respectively. U represents μmol/h.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/63e1195958dab488dbd02e01.png"},{"id":52083537,"identity":"508674e9-2663-490f-8cfe-cc579b7e89e2","added_by":"auto","created_at":"2024-03-06 11:40:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":124867,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of β-1,3-glucanase mode of action on laminarin oligosaccharides. The enzyme was sourced from the RWASA2 infested Tugela (S), Tugela \u003cem\u003eDn1\u003c/em\u003e(MR), and Tugela \u003cem\u003eDn5\u003c/em\u003e (R) cultivars at 3 dpi. Laminarin oligosaccharides were represented by L5, L4, L3 and L2, which were equivalent to Laminaripentaose (LAM5), Laminaritetraose (LAM4), Laminaritriose (LAM3), and Laminaribiose (LAM2), respectively. The red arrow shows the glucose (GLU), and the blue arrow shows the profiles of the enzyme without the oligosaccharides.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/33e5fe1248d54dc6247ac0e4.png"},{"id":61596486,"identity":"2a80ef20-1b31-4d50-81a4-491da3cc4e3a","added_by":"auto","created_at":"2024-08-01 17:27:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1772794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3968206/v1/c229d463-6089-4a55-857b-9f99350e2510.pdf"}],"financialInterests":"","formattedTitle":"Functional characterization of cell wall-associated β-glucanases and peroxidase induced during wheat-Diuraphis noxia inteactions.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWheat plants infested by Russian wheat aphids (RWA) induce a cascade of defence responses, including the hypersensitive response (HR). These defence responses involve synthesising distinct pathogenesis-related (PR) proteins activated upon plant invasion by biotic stressors (Mohase and van der Westhuizen, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e; Manghwar et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The PR proteins, such as β-1,3-glucanase (EC. 3.2.1.39) and peroxidase (POD: EC 1.11.1.7) were significantly induced to higher levels in the resistant cultivars compared to the susceptible cultivars infested with RWA (Mohase and van der Westhuizen, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e; Mohase and van der Westhuizen, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2002b\u003c/span\u003e; Moloi and van der Westhuizen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The suggested physiological functions of β-1,3-glucanase and POD in the resistant cultivars included cell wall modification and reactive oxygen species (ROS) quenching at the cell wall and apoplastic regions during RWA infestation (Mohase and van der Westhuizen, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e; Botha and Matsiliza, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Moloi and van der Westhuizen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mafa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Walker, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe β-1,3-glucanases, PODs and chitinases (EC 3.2.1.14) are part of the most studied PR proteins during wheat-RWA interaction (Mohase and van der Westhuizen, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e; Moloi and van der Westhuizen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Farvardin et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bhardwaj et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Other PR proteins involved in plant defence reported by Appu et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), include chitosanases (EC 3.2.1.132), phenylalanine ammonia-lyase (EC 4.3.1.5) and polyphenol oxidase (EC 1.10.3.2). In the cases of β-1,3-glucanase and POD, most studies were performed using the apoplastic fluid extracted from wheat samples infested with RWA. However, there is a lack of information regarding the synergistic effect of the β-1,3-glucanase and POD on plant cell wall modification during wheat-RWA interaction. Thus, herein, we attempted to elucidate the physiological defensive roles of POD and β-1,3-glucanase on cell wall strengthening during wheat-RWA interaction.\u003c/p\u003e \u003cp\u003eBased on the Carbohydrate-Active enZymes (CAZymes) Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cazy.org\u003c/span\u003e\u003cspan address=\"http://www.cazy.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), β-1,3-glucanases belong to the glycoside hydrolase (GH) family 17. They are classified into two types, i.e., \u003cem\u003eendo\u003c/em\u003e-β-1,3-glucanases (EC 3.2.1.39) that cleave the β-1,3-linkages inside the β-glucan chain at random sites, while \u003cem\u003eexo\u003c/em\u003e-β-1,3-glucanases (EC 3.2.1.58) cleave the β-1,3-linkages from the nonreducing end of the β-glucan chain (Rajninec et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Both \u003cem\u003eendo-\u003c/em\u003e and \u003cem\u003eexo\u003c/em\u003e-acting β-1,3-glucanases degrade β-1,3-glucan efficiently, which regulates callose accumulation in the resistant cultivar. Other studies argued that by regulating callose, these enzymes produce β-1,3-glucan oligosaccharides (Forslund et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Rajninec et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The β-1,3-glucan oligosaccharides were also referred to as DAMPs (damage-associated molecular patterns), which induced pattern triggered immune (PTI) responses in plants (Forslund et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Rajninec et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rebaque et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe β-1,3-glucanase upregulation, alone or in collaboration with other proteins, contributed to plant resistance to RWA infestation (Wang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is important to note that there are distinct types of beta-glucan polysaccharides in terrestrial plants; they include β-1,3-glucan; β-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4 glucan, and β-1,4-glucan (Rebaque et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, it is not clear if the upregulated β-1,3-glucanase during RWA-wheat infestation is only active on one type of β-glucan or if it can also hydrolyse other types. In a study conducted by Manghwar et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), large contents of β-1,3-glucanase coding genes were expressed in resistant wheat and maize cultivars infected with \u003cem\u003eBipolaris sarokiniana\u003c/em\u003e, relative to controls. This observation suggests that β-1,3-glucanase is indeed significant for a plant to survive against biotic stress (including RWA infestation).\u003c/p\u003e \u003cp\u003ePeroxidases participate in plant defence-related responses by strengthening plant cell walls through lignification, formation of intermolecular cross-links, and suberin formation (Moloi and van der Westhuizen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mnich et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mafa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Peroxidase is an important biochemical marker in pest-plant interactions because its activity increases in resistant cultivars during RWA infestation (Xu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The increased peroxidase activity in resistant wheat after RWA infestation was important for the activated defence responses (Mafa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Peroxidase can either induce defensive responses or reduce the harmful effects of oxidative stress in the cell wall region (Mnich et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Appu et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe preceding sections of the introduction demonstrate that a lot of work has been done on the β-1,3-glucanase and POD found in the cytoplasmic and apoplastic regions. However, there are fewer or no studies that distinguished β-1,3-glucanase and POD associated with the cell wall to elucidate their synergistic role on the cell wall modification. Hence, the current study attempted to comprehensively characterize the physicochemical properties of the plant cell wall associated POD and β-1,3-glucanase in wheat cultivars infested with RWASA2.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eTugela, Tugela \u003cem\u003eDn1\u003c/em\u003e, and Tugela \u003cem\u003eDn5\u003c/em\u003e seeds were supplied by the Agricultural Research Council\u0026ndash;Small Grain (ARC-SG) Institute, Bethlehem, South Africa. A fresh colony of RWASA2 was developed on the susceptible PAN 3118 wheat cultivar in the glasshouse at UFS, Bloemfontein Campus (29\u0026deg;6\u0026rsquo;31.94\u0026rsquo;\u0026rsquo; S; 26\u0026deg;11\u0026rsquo;18.95\u0026rsquo;\u0026rsquo; E). The commercial barley \u003cem\u003eendo\u003c/em\u003e-β-1,3-glucanase enzyme was purchased from Megazyme (Wicklow, Ireland). The Curdlan (β-1,3-glucan), barley mixed linked β-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-glucan (MLG), AZO-carboxymethyl cellulose (CMC), and Laminarin oligosaccharides (Laminaripentaose, Laminaritetraose, Laminaritriose, and Laminaribiose) were purchased from Megazyme (Wicklow, Ireland). The commercial guaiacol substrate was purchased from Sigma (Johannesburg, South Africa). All the chemicals for buffer preparations and all analytical chemicals used in the study were purchased from Sigma (Johannesburg, South Africa), unless stated otherwise.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth conditions\u003c/h2\u003e \u003cp\u003eThe trial was laid as a split-plot design, with infestation as the main plot and cultivars sub-plots and replicated four times. Plants were cultivated according to Mafa et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, 250 seeds of susceptible Tugela, moderately resistant Tugela \u003cem\u003eDn1\u003c/em\u003e and resistant Tugela \u003cem\u003eDn5\u003c/em\u003e cultivars were germinated in separate Petri dishes, respectively. Germination conditions were as follows: 5 mL of distilled water was added to each Petri dish containing seeds. Petri dishes were sealed with a parafilm and incubated in a dark germination chamber at 25\u0026deg;C for 2 days. Tugela seeds differed from other seeds and took 7 days to germinate. The germinated seeds of each cultivar were transplanted in 15 cm pots containing 1:1 soil and peat moss (Mikskaar Professional Substrate) under controlled glasshouse conditions at the UFS, Bloemfontein Campus, South Africa. The plants were grown under a natural photoperiod and temperature regimes were 18\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C during night and day, respectively. The plants were irrigated every three days using filtered tap water and fertilised with 2 g/L Multifeed classic\u0026trade; [5.2.4 (43)] 14 days after germination. The plants were enclosed in cages covered with nets and allowed to grow up to the second leaf stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRussian wheat aphid (RWASA2) infestation\u003c/h2\u003e \u003cp\u003eTugela, Tugela \u003cem\u003eDn1\u003c/em\u003e, and Tugela \u003cem\u003eDn5\u003c/em\u003e wheat cultivars were infested with RWASA2 according to Jimoh \u003cem\u003eet al\u003c/em\u003e. (2013) and Mohase and Taiwe (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Twenty freshly harvested adult RWASA2 clone aphids were used to infest each plant at the three-leaf growth stage. The uninfested controls and infested test plants were kept in separate rooms. All plants were enclosed in cages covered with nylon nets (315 \u0026micro;m). There were two harvesting regimes: short-term feeding (1, 2, and 3 days) and long-term feeding (7 and 14 days). At each harvesting time ten randomly selected leaves (second and third) of each cultivar were cut at the base, wrapped in moist paper towel, immediately placed on ice and transferred to the laboratory for intercellular wash fluid (IWF) extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of cell wall associated β-1,3-glucanase and Peroxidase (POD)\u003c/h2\u003e \u003cp\u003eThe harvested leaves were cut into 7 cm pieces. To remove any cytosolic contamination from the cut ends, the leaf pieces were rinsed twice in distilled water (Van der Westhuizen et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1998a\u003c/span\u003e) and submerged into a 50 mM Tris-HCl extraction buffer (pH 7.8) in a thick-walled glass tube. The leaf pieces were impregnated with extraction buffer following a 5-minute infiltration under vacuum created by a water jet pump. Thereafter, leaves were removed from the glass tube, dried with a paper towel, placed vertically into pre-cooled centrifuge tubes fitted with a perforated disk, and centrifuged at 500 \u0026times;\u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C (Mafa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The extraction procedure was repeated, and the collected supernatant was divided into aliquots and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. However, the IWF was not used in the current study, rather was extracted to ensure the enzymes studied were not apoplastic enzymes. The leaf tissue left after IWF extraction was used to extract the total protein, which was used as a source of cell wall-bound β-1,3-glucanase and POD. Leaf residues were crushed to a fine powder in liquid nitrogen using a mortar and pestle. About 200 mg of the powder was extracted with 4 mL extraction buffer (50 mM sodium phosphate, pH 6.0, containing 1% (\u003cem\u003ew/v\u003c/em\u003e) polyvinylpolypyrrolidone (PVPP). The suspension was allowed to rest for 3 min and then centrifuged at 10 000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 15 min. The supernatant (total proteins extracts) was collected into 2 mL Eppendorf tubes and used as source of the β-1,3-glucanase and POD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProtein concentration quantification\u003c/h2\u003e \u003cp\u003eThe protein concentration of total protein extracts was determined according to Bradford (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). Briefly, 10 \u0026micro;L of the enzyme extract was mixed with 190 \u0026micro;L Bradford reagent (BioRad) in a microplate well and incubated at 25\u0026deg;C for 20 min. The protein concentration was determined by measuring the absorbance at 595 nm using a microplate reader (Anthos, Zenyth 3100). Bovine serum albumin (BSA) was used as a suitable standard to develop a calibration curve with concentrations ranging between 0.05 and 1 mg/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eβ-1,3-glucanase activity assay\u003c/h2\u003e \u003cp\u003eThe enzyme activity of the cell wall-bound β-1,3-glucanase extracted from the leaves was determined by measuring the amount of reducing sugars released from the hydrolysis of 0.5% (\u003cem\u003ew/v\u003c/em\u003e) mixed linked β-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-glucan (MLG) and 0.4% (\u003cem\u003ew/v\u003c/em\u003e) β-1,3-glucan substrates using a modified method of Miller (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1959\u003c/span\u003e) and Mafa et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The reaction mixture contained 200 \u0026micro;L enzyme extract (protein concentrations were kept between 0.02 to 0.09 mg/mL in all reactions, unless stated otherwise), and 300 \u0026micro;L of the substrate dissolved in 50 mM sodium citrate buffer (pH 5). Each reaction mixture was incubated at 37\u0026deg;C for 24 h and the reaction was terminated by heating at 100\u0026deg;C for 5 min followed by centrifugation at 10 000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 10 min. About 300 \u0026micro;L supernatant containing the reaction hydrolysate was mixed with 600 \u0026micro;L 3,5-Dinitrosalicylic acid (DNS) reagent at a 1:2 ratio; boiled at 100\u0026deg;C for 5 min and allowed to cool at room temperature. The absorbance of the samples was measured at 540 nm using a spectrophotometer (GENESYS 120 UV\u0026ndash;Visible Spectrophotometer). Glucose was used as a standard to establish a standard curve used in determining the enzyme activity, expressed as \u0026micro;mol glucose/h/mg protein. One unit of β-1,3-glucanase activity was defined as the amount of enzyme that released 1 \u0026micro;mol of glucose/h from the substrate under the described conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePOD activity assay\u003c/h2\u003e \u003cp\u003eA modified method of Zieslin and Ben-Zaken (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) was used to determine cell wall-bound POD activity in the wheat cultivars. The assay mixture consisted of 970 \u0026micro;L of 5 mM guaiacol, 30 \u0026micro;L of 1% (\u003cem\u003ev/v\u003c/em\u003e) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and 30 \u0026micro;L enzyme extract. The reaction was performed at 25\u0026deg;C, and the change in absorbance was monitored at 470 nm using the kinetic mode of a spectrophotometer (GENESYS 120 UV\u0026ndash;Visible Spectrophotometer). Peroxidase activity was determined using the molar extinction coefficient of guaiacol (26.6 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and expressed in \u0026micro;mol tetra-guaiacol/minute/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical characterization\u003c/h2\u003e \u003cp\u003eBiochemical assays for cell wall-bound β-1,3-glucanase and POD induced in the wheat cultivars were performed to determine the pH and temperature optima, and thermostability. Based on the data analysis, the 3 dpi samples were selected to minimize bias between different cultivars\u0026rsquo; responses to RWA infestation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePOD characterization\u003c/h2\u003e \u003cp\u003eEach reaction consisted of 30 \u0026micro;L enzyme extract, 1% (\u003cem\u003ev/v\u003c/em\u003e) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 5 mM guaiacol. The formation of the products was measured at 470 nm using the kinetic mode of the of the spectrophotometer (GENESYS 120 UV\u0026ndash;Visible Spectrophotometer). The optimum pH studies for POD activity were conducted at 25\u0026deg;C using guaiacol dissolved in different 50 mM buffers, i.e., sodium citrate buffer with a pH of 4 and 5; sodium phosphate buffer with a pH of 6 and 7; and Tris-HCL buffer with a pH of 8 and 9. For optimum temperature tests, assays were conducted at 25, 30, 40 and 50\u0026deg;C, respectively. The reactions were conducted for 5 min using guaiacol dissolved in 50 mM sodium citrate buffer (optimum pH). For thermostability studies, the reactions were conducted by incubating the enzyme at 37, 50 and 70\u0026deg;C for 30 min, and reactions were conducted as described for temperature and pH optima assays. All the reactions were conducted in quadruplicates. Activity was expressed as POD relative activity using percentages and specific activity (U/mg protein).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eβ-1,3-glucanase characterisation\u003c/h2\u003e \u003cp\u003eThe MLG and β-1,3-glucan substrates were hydrolysed by the β-1,3-glucanase and the production of total reducing sugars was measured by DNS reagents as elaborated in section 3.3.7 above. The optimum pH for the enzymes was determined by adding 200 \u0026micro;L of the enzyme extract to 300 \u0026micro;L substrate dissolved in different buffers (50 mM sodium citrate pH 4 \u0026amp; 5; sodium phosphate pH 6 \u0026amp; 7; Tris-HCL pH 8 \u0026amp; 9) and incubating the reaction at 37\u0026deg;C for 24 h. For determining optimum temperature, the reactions were incubated at 25, 30, 40, and 50\u0026deg;C. For thermostability assays, the stability of the extracted enzyme was determined by first incubating for 30 min at 25, 37, 50, and 70\u0026deg;C followed by the reaction conducted as described above. All the reactions were conducted in triplicates. Activity was expressed in relative activity using percentages and specific activity (U/mg protein).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eβ-1,3-glucanase mechanism of action assays\u003c/h2\u003e \u003cp\u003eSubstrate specificity was assessed to determine further if the β-1,3-glucanase induced by RWASA2 infestation in the wheat cultivars hydrolysed β-1,3-glucan (CM-curdlan), MLG (from barley) and β-1,4-glucan (AZO-CM-Cellulose) substrates. The mechanism of action was determined under optimal assay conditions (25 \u0026amp; 40\u0026deg;C, pH 5.0) using 200 \u0026micro;L enzyme extract, 300 \u0026micro;L of the substrate [0.1% (\u003cem\u003ew/v\u003c/em\u003e) β-1,4-glucan, 0.4% (\u003cem\u003ew/v\u003c/em\u003e) β-1,3-glucan, or 0.5% (\u003cem\u003ew/v\u003c/em\u003e) MLG] dissolved in 50 mM sodium citrate buffer (pH 5). Reactions were incubated at 25 and 40\u0026deg;C for 8 h, followed by termination by heating at 100\u0026deg;C. Proceeding reaction steps for β-1,3-glucan and MLG were conducted as described in section 3.3.7 above. For β-1,4-glucan, after terminating the reaction at 100\u0026deg;C, the mixture was diluted with 800 \u0026micro;L absolute ethanol followed by centrifugation at 10 000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 10 min. After centrifugation, the absorbance was measured at 590 nm. The β-1,3-glucanase activity on β-1,4-glucan was calculated using the manufacturer\u0026rsquo;s (Megazyme) procedure and expressed in Units/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetermining β-1,3-glucanase mode of action\u003c/h2\u003e \u003cp\u003eThe β-1,3-glucanase mode of action was assayed with laminarin-oligosaccharides (LAMs) with degree of polymerization (DP) between 5 and 2. Thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), and glucose oxidase peroxidase (GOPOD) were used to determine the DP required for β-1,3-glucanase to hydrolyse the substrate, to quantify the concentration of the oligosaccharides with DP between 5 and 2 and glucose, respectively (Mafa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mafa et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The total protein extract was concentrated with 10 kDa centrifuge concentrating membrane filters, and it was used as a concentrated β-1,3-glucanase sourced from samples at 3 dpi. Enzyme extracts from RWASA2-infested wheat (20 \u0026micro;L of concentrated or non-concentrated enzyme) were incubated at 40\u0026deg;C for 16 h with 10 mg/mL of Laminaripentaose (LAM5), Laminaritetraose (LAM4), Laminaritriose (LAM3), and Laminaribiose (LAM2) (Megazyme), dissolved in 50 mM sodium citrate (pH5). The reaction was terminated by boiling for 5 min before spotting the samples on the TLC.\u003c/p\u003e \u003cp\u003eThe soluble oligosaccharides (15 \u0026micro;L) reaction mixtures were spotted on the silica plate [TLC Silica gel 60 F\u003csub\u003e254,\u003c/sub\u003e HPTLC plate (Merck, Darmstadt, Germany)]. TLC was conducted using the mobile phase of n-butanol: acetic acid: water (2:1:1 \u003cem\u003ev/v/v\u003c/em\u003e). The plate was allowed to develop for 2 h. The plate was removed from the mobile phase, dried, and stained with 0.3% (\u003cem\u003ew/v\u003c/em\u003e) Naphthol (Merck, Germany) in 95% (\u003cem\u003ev/v\u003c/em\u003e) ethanol using 5% (\u003cem\u003ev/v\u003c/em\u003e) sulfuric acid (Merck, Darmstadt, Germany) followed by heating at 100\u0026deg;C for 7 min. The visible blue-violet spots were documented.\u003c/p\u003e \u003cp\u003eThe concentration of the hydrolysate (oligosaccharides) produced during hydrolysis of LAMs was determined with the LC-MS as described by Mafa et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hydrolysates were analysed using an ABSCIEX 4000 QTRAP hybrid triple quadrupole ion trap mass spectrometer with a Shimadzu high-performance liquid chromatography (HPLC) stack as a front end. Analyst 1.5 (AB SCIEX) software was used to perform all acquisition and processing of the data. Briefly, twenty microliters of each sample were separated on a C18 (Carbohydrate 4.6 \u0026times; 250 mm, Agilent) column at 500 \u0026micro;L/min flow rate over 10 min using a water (solvent A) and acetonitrile (solvent B) gradient from 100% B to 60% B, followed by column re-equilibration steps with a total run time of 20 min. Eluting analytes were ionised in negative electrospray mode in the TurboV ion source with a 400\u0026deg;C-heater temperature to evaporate the excess solvent, 30 psi nebuliser gas, 30 psi heater gas and 20 psi curtain gas. Declustering potential was set at 350 V. Mass spectrometer was used to analyse the eluting analytes in a Q1 scan mode ranging from 150 Da to 1000 Da with a dwell time of 3 s. The glucose concentration produced by laminarin hydrolysis was further analysed using GOPOD reagent following Megazyme instructions. The absorbance was measured at 510 nm in a fixed mode of the spectrophotometer (GENESYS 120 UV\u0026ndash;Visible Spectrophotometer).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData collection and analysis\u003c/h2\u003e \u003cp\u003eAll experiments were randomised to avoid bias during infestation or sample collection, and analysis was conducted in quadruplicates. The values in the graphs and tables generated from the collected experimental data represent the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation unless stated otherwise. Microsoft Excel was used to analyse all the data and to generate graphs. Statistical analysis was performed with TIBCO Statistica (version 13.1), whereby multifactorial Analysis of variance (ANOVA) was performed to test for significance between treatments and the Fisher's LSD was used to test for homogenous groups at alpha value of 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003ePeroxidase activity determination\u003c/h2\u003e\n \u003cp\u003ePeroxidase activity of the control relative to RWASA2-infested resistant Tugela \u003cem\u003eDn\u003c/em\u003e5 and moderately resistant Tugela \u003cem\u003eDn\u003c/em\u003e1 was not significantly different at 1 dpi, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. It is only in the susceptible Tugela where the POD activity in the infested was higher relative to the control. At 2, 3 and 7 dpi, RWASA2 infestation induced higher POD activity in all three cultivars than in the control treatments. In the moderately resistant Tugela \u003cem\u003eDn\u003c/em\u003e1 and resistant Tugela \u003cem\u003eDn\u003c/em\u003e5, RWASA2 infestation induced significantly higher POD activity relative to the control treatment at 14 dpi, showing a delayed response in the resistant cultivar. However, the opposite was observed in the susceptible Tugela cultivar (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), where POD activity was repressed by RWASA2 infestation relative to the control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e\u0026beta;-1,3-glucanase activity\u003c/h2\u003e\n \u003cp\u003eRWASA2 infestation significantly induced higher \u0026beta;-1,3-glucanase activity in the resistant Tugela \u003cem\u003eDn5\u003c/em\u003e and moderately resistant Tugela \u003cem\u003eDn1.\u003c/em\u003e Lower activity levels were induced in the susceptible Tugela. Both RWASA2 infested and control cultivars showed that high \u0026beta;-1,3-glucanase activity induced in the cell wall of all three cultivars significantly increased over the 14 days post infestation (dpi) during MLG hydrolysis (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The activity of the \u0026beta;-1,3-glucanase extracted from Tugela did not show any specific pattern during the hydrolysis of MLG substrate and was significantly reduced at 1 and 14 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The activity of the enzyme extracted from Tugela \u003cem\u003eDn1\u003c/em\u003e significantly increased at all time points, and was gradually increasing over time, except at 2 dpi. However, \u0026beta;-1,3-glucanase activity was significantly reduced relative to control at 1 and 2 dpi in Tugela \u003cem\u003eDn5\u003c/em\u003e during the MLG hydrolysis. The activity of this enzyme seemed to be delayed showing significant increase in the samples infested for 7 and 14 days (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eWhen the curdlan (\u0026beta;-1,3-glucan) was hydrolysed by \u0026beta;-1,3-glucanase extracted from susceptible Tugela, there were no significant differences between control and infested samples at 2 and 7 dpi, followed by significant increase activity at 3 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, it is important to note that at 1 and 14 dpi the \u0026beta;-1,3-glucanase was significantly reduced compared to controls in the susceptible cultivar. The RWASA2 infested Tugela \u003cem\u003eDn\u003c/em\u003e1, and control samples had comparable \u0026beta;-1,3-glucanase activity levels, but at 3 and 7 dpi the infested cultivar samples had significantly higher \u0026beta;-1,3-glucanase activity (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). In contrast, this enzyme displayed significantly increased activity in the Tugela \u003cem\u003eDn5\u003c/em\u003e samples from 3 to 14 dpi compared to control samples (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). Overall, the results show that there are two distinct enzymes, which catalyse the MLG and \u0026beta;-1,3-glucan substrates such as curdlan independently. It was interesting to observe that both enzymes were significantly induced in the moderate and resistant Tugela cultivars at 3 to 14 dpi, while both enzymes\u0026rsquo; activities were repressed in the susceptible Tugela cultivar after 3 dpi.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eBiochemical characterization\u003c/h2\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003ePOD characterization\u003c/h2\u003e\n \u003cp\u003eThe biochemical conditions for POD in the susceptible Tugela, moderately resistant Tugela \u003cem\u003eDn1\u003c/em\u003e, and resistant Tugela \u003cem\u003eDn5\u003c/em\u003e were assessed using guaiacol substrate in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and the results displayed in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e exhibited higher activities in acidic conditions. RWASA2 infestation induced significantly higher POD activity in Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e compared to control samples and higher activity was recorded at pH 6 and pH 5 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and C). The POD activity in the control samples was slightly higher than in infested Tugela samples (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The POD activity was significantly reduced from neutral to alkaline conditions. For instance, all samples lost more than 40 and 50% relative activity at pH 7 and 8, respectively. At pH 9, this enzyme lost over 90% relative activity in infested or controls of the three cultivars. The specific activity shows that POD activity levels were lower in Tugela (\u0026lt;\u0026thinsp;0.8 U/mg protein), while the enzyme activity levels were more than 1.2 U/mg protein in Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e samples. The significantly higher POD activity in moderately resistant Tugela \u003cem\u003eDn1\u003c/em\u003e and resistant Tugela \u003cem\u003eDn5\u003c/em\u003e samples post infestation at acidic pH range demonstrate that this enzyme was associated with the cell wall reinforcements.\u003c/p\u003e\n \u003cp\u003eThe optimum temperature of the POD enzyme was assessed in both the RWASA2-infested and control cultivars. Relative activity of this enzyme displayed an increasing trend from 25 to 50\u0026deg;C (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), but the controls of Tugela and Tugela \u003cem\u003eDn5\u003c/em\u003e did not show any different activity between 25 and 30\u0026deg;C. Infested Tugela \u003cem\u003eDn5\u003c/em\u003e sample displayed significantly increased POD activity from 25 to 50\u0026deg;C, displaying 100% relative activity at 50\u0026deg;C. Similarly, RWASA2 infestation in Tugela \u003cem\u003eDn1\u003c/em\u003e induced higher activity compared to control at all tested temperatures, with the maximum relative activity at 50\u0026deg;C. Tugela also had maximum activity for both treatments at 50\u0026deg;C, but the infestation reduced the POD activity by about 5% compared to the control. Once again, POD activity was significantly increased post RWA infestation in the Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e samples, while POD extracted from Tugela control and infested sample did not show any differences (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD, E and F).\u003c/p\u003e\n \u003cp\u003eThe thermostability assay was assessed by incubating the enzyme extracts at 37, 50, and 70\u0026deg;C for 30 min. Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e showed that POD extracted from RWASA2 infested Tugela \u003cem\u003eDn1\u003c/em\u003e samples retained maximum (100%) relative activity at 37\u0026deg;C and gradually lost activity as the temperature increased. The enzyme retained over 80% activity at 50\u0026deg;C. In addition, RWASA2 infested Tugela and Tugela \u003cem\u003eDn5\u003c/em\u003e samples significantly reduced the POD activity compared to the control, suggesting that POD extracted from this samples was not thermo-tolerant compared to the control (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and C). At 70\u0026deg;C, POD relative activity was lost in infested or control samples of the three cultivars, indicating that POD is a mesophilic enzyme that cannot tolerate temperatures up to 70\u0026deg;C.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e\u0026beta;-1,3-glucanase characterization\u003c/h2\u003e\n \u003cp\u003eThe optimum conditions of \u0026beta;-1,3-glucanase activity from Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e, and Tugela \u003cem\u003eDn5\u003c/em\u003e were assessed using MLG and \u0026beta;-1,3-glucan (Curdlan) substrates (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). RWASA2 infestation induced more enzyme activity relative to control in all three cultivars under acidic pH conditions. The \u0026beta;-1,3-glucanase showed higher enzyme activity in acidic conditions, with pH 5 showing maximum enzyme activity in all three cultivars and over 90% relative activity induced at pH 4 when MLG was used as a substrate (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, B and C). At neutral pH ranges this enzyme showed a significantly decreased relative activity (retaining between 50% and 40% activity) in Tugela and Tugela \u003cem\u003eDn1\u003c/em\u003e, while it lost more than 60% relative activity in Tugela \u003cem\u003eDn5\u003c/em\u003e samples from pH 6 to pH 9. In addition, the specific activity showed that Tugela \u003cem\u003eDn5\u003c/em\u003e has higher levels of \u0026beta;-1,3-glucanase activity (reaching more than 20 U/mg protein) compared to moderately resistant or susceptible cultivars.\u003c/p\u003e\n \u003cp\u003eWhen the \u0026beta;-1,3-glucanase pH optima studies were conducted using the Curdlan, the relative activity profile of all three cultivars differed from the one displayed when MLG was used as a substrate. It is also worth noting that infestation induced \u0026beta;-1,3-glucanase activity in Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e over acidic pH range (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, E and F). In addition, the enzyme showed the optimum pH at 5 in the RWASA2 infestation samples and controls. The enzyme lost more than 60% relative activity in the Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e from pH 8 and pH 9, while Tugela samples lost more than 70% at the same alkaline pH range. The results in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrated that the enzymes hydrolysed curdlan and MLG were two different enzymes. This observation supports the earlier specific activity result, suggesting that this enzyme could be MLG specific \u0026beta;-glucanase or two distinctly different \u0026beta;-1,3-glucanase isozymes.\u003c/p\u003e\n \u003cp\u003eThe optimum temperature of \u0026beta;-1,3-glucanase enzyme extracted from RWASA2-infested wheat cultivars was assessed using temperatures ranging from 25 to 50\u0026deg;C. Figure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e results showed that during the hydrolysis of MLG substrate, the \u0026beta;-1,3-glucanase displayed maximum enzyme activities at 25\u0026deg;C in Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e samples. The enzymes retained more than 90% relative activities in Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e samples at 40 and 50\u0026deg;C, but about 80% relative activity was retained in Tugela samples. Therefore, 25\u0026deg;C was the optimum temperature for \u0026beta;-1,3-glucanase on the MLG substrate. In addition, the specific activity levels of the \u0026beta;-1,3-glucanase in the Tugela \u003cem\u003eDn5\u003c/em\u003e were significantly high compared to the control (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Also, \u0026beta;-1,3-glucanase specific activity levels in Tugela \u003cem\u003eDn5\u003c/em\u003e was 2-fold higher than those displayed by Tugela \u003cem\u003eDn1\u003c/em\u003e and 4-fold higher than those displayed by Tugela.\u003c/p\u003e\n \u003cp\u003eDuring the hydrolysis of curdlan substrate, \u0026beta;-1,3-glucanase showed an optimum temperature at 40\u0026deg;C for the three wheat cultivars. At 50\u0026deg;C the enzyme retained more than 80% relative activity in the RWASA2-infested Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e samples (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD, E and F). Once again, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e \u0026beta;-1,3-glucanase specific activities were significantly higher in the infested samples compared to controls. However, no significant differences were observed between infested Tugela and control samples. The enzyme specific activities were similar in Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e (reaching values around 7.5 U/mg protein). At 40\u0026deg;C, the RWASA2 infested moderately resistant and resistant cultivars showed 3-fold higher specific activity compared to controls. The results of Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e displayed two different catalytic patterns when the enzyme acted on the MLG and curdlan, suggesting there were two different enzymes or \u0026beta;-1,3-glucanase isozymes or MLG specific glucanase and \u0026beta;-1,3-glucanase.\u003c/p\u003e\n \u003cp\u003eThe thermostability assay was conducted by incubating the enzyme extract at 25, 37, 50, and 70\u0026deg;C for 30 min. The percentage relative activity results showed that \u0026beta;-1,3-glucanase enzyme extracted from RWASA2-infested Tugela \u003cem\u003eDn5\u003c/em\u003e and Tugela \u003cem\u003eDn1\u003c/em\u003e samples was significantly thermostable compared to the control (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB and C) hydrolysing MLG. In addition, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e infested with RWASA2 induced the highest \u0026beta;-1,3-glucanase specific activity at 25\u0026deg;C during the hydrolysis of MLG. Figure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB and C results showed that at 50 and 70\u0026deg;C the \u0026beta;-1,3-glucanase extracted from Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e retained over 80% relative activity during the hydrolysis of MLG. These results confirm that the enzymes catalysing the MLG substrates were thermostable in resistant cultivars.\u003c/p\u003e\n \u003cp\u003eIn contrast, the thermostability assay of \u0026beta;-1,3-glucanase extracted from the three wheat cultivars showed that it is a mesophilic enzyme during the hydrolysis of \u0026beta;-1,3-glucan substrate. At 37\u0026deg;C, the enzyme extracted from Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e, and Tugela \u003cem\u003eDn5\u003c/em\u003e infested with RWASA2 displayed over 80% relative activity. The \u0026beta;-1,3-glucanase activity increased as the temperature increased to 50\u0026deg;C in all three cultivars. Once again, RWASA2 infested Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e induced significantly higher enzymatic activities relative to controls. Also, results in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD, E and F showed that \u0026beta;-1,3-glucanase displayed significant reduction in enzyme activity at 70\u0026deg;C in the infested and controls samples of the three wheat cultivars. These findings show that \u0026beta;-1,3-glucanase enzyme that hydrolyses the curdlan (\u0026beta;-1,3-glucan) substrate is not a thermostable enzyme, but a mesophilic enzyme, while the MLG specific enzyme was thermostable.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e\u0026beta;-1,3-glucanase mechanism of action\u003c/h2\u003e\n \u003cp\u003eMechanism of action is a study that explains the glycosidic linkages required for the enzyme to be active. The previous \u003cspan class=\"InternalRef\"\u003eresults\u003c/span\u003e sections showed that the enzymes acting on MLG and curdlan substrates differed. This observation warranted further investigation to probe if the \u0026beta;-1,3-glucanase has similar activity on glucan substrates with different glycosidic linkages (i.e., MLG, curdlan and CMC substrates) under optimum condition. Figure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA shows \u0026beta;-1,3-glucanase activity on \u0026beta;-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-glucan substrate. The enzyme from Tugela \u003cem\u003eDn5\u003c/em\u003e had higher activity than the one extracted from Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela, showing more activity at 25\u0026deg;C than at 40\u0026deg;C. In addition, \u0026beta;-1,3-glucanase extracted from Tugela showed significantly lower activity levels during hydrolysis of curdlan (\u0026beta;-1,3-glucan) substrate. In contrast, Tugela \u003cem\u003eDn1\u003c/em\u003e displayed the highest enzyme activity at 40\u0026deg;C during curdlan hydrolysis (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). The RWASA2-infested resistant Tugela \u003cem\u003eDn5\u003c/em\u003e \u0026beta;-1,3-glucanase activity levels were induced 3-fold more during the hydrolysis of the substrate with \u0026beta;-1,3-glycosidic linkages compared to when it hydrolysed the substrate with \u0026beta;-1,4-glycosidic linkages; and 2-folds more activity compared to when it hydrolysed the \u0026beta;-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-glycosidic linkage. These findings support our claim that there are two different \u0026beta;-1,3-glucanase isozymes or \u0026beta;-1,3-glucan and MLG specific enzymes, which are significantly induced by RWASA2 in Tugela \u003cem\u003eDn5\u003c/em\u003e.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eHydrolytic patterns of \u0026beta;-1,3-glucanase\u003c/h2\u003e\n \u003cp\u003eLaminari-oligosaccharides which included laminaripentaose (LAM5), laminaritetraose (LAM4), laminaritriose (LAM3), and laminaribiose (LAM2) were hydrolysed with \u0026beta;-1,3-glucanase extracted from the RWASA2 infested Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5;\u003c/em\u003e the reaction was conducted for 16 h and the hydrolysate was analysed with TLC. The \u0026beta;-1,3-glucanase without the oligosaccharides (control) showed a single band of monosaccharide in all three cultivars, corresponding to the glucose molecule. The \u0026beta;-1,3-glucanase extracted from Tugela \u003cem\u003eDn5\u003c/em\u003e hydrolysed the LAM5 to LAM2 efficiently, producing glucose (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). LAM5 hydrolysis produced higher amounts of glucose, followed by LAM3 and LAM2. The enzyme extracted from Tugela \u003cem\u003eDn1\u003c/em\u003e samples was the second most effective in hydrolysing LAM5, LAM4, LAM3, and LAM2 into glucose. It also produced a similar oligosaccharide pattern to the enzyme extracted from the resistant Tugela \u003cem\u003eDn5\u003c/em\u003e cultivar. The least effective enzymes were extracted from Tugela samples, and they showed higher activity on LAM5 compared to shorter oligosaccharides (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eLaminarin-oligosaccharides hydrolysates analysed with LC-MS\u003c/h2\u003e\n \u003cp\u003eThe laminarin-oligosaccharides with the DP between 5 and 2 were hydrolysed with \u0026beta;-1,3-glucanase extracted from the wheat cultivars infested with RWASA2 for 3 days and the reaction products were quantified by LC-MS and GOPOD reagents. Table 3.1 showed that the non-concentrated enzymes could hydrolyse the LAM5 into glucose (green colour of the heat map), followed by a moderate concentration of LAM4 (yellow colour). The heat map shows that the LAM3 and LAM2 produced during the LAM5 hydrolysis were in trace concentration (red colour). The non-concentrated enzyme extracted from Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e did not hydrolyse the substrate oligosaccharides efficiently, leaving them with high concentrations ranging between 183 and 122 mg/L (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e green colour). In contrast, Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results of the concentrated \u0026beta;-1,3-glucanase sourced from the Tugela \u003cem\u003eDn5\u003c/em\u003e, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela at 3 dpi. Tugela \u003cem\u003eDn1\u003c/em\u003e \u0026beta;-1,3-glucanase was highly effective during the hydrolysis of Laminarin-oligosaccharides with DP between 5 and 2. This enzyme showed the highest activity on the longer oligosaccharides (LAM5 and LAM4), leaving about 9.6 and 15.9 mg/L, respectively. However, it showed moderate activity during the hydrolysis of shorter oligosaccharides (LAM3 and LAM2), leaving about 40.2 and 66.8 mg/L, respectively. The Tugela \u003cem\u003eDn1\u003c/em\u003e \u0026beta;-1,3-glucanases produced a higher glucose concentration, followed by tetraose and biose during the hydrolysis of LAM5 to LAM2 (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The concentrated enzyme extracted from Tugela \u003cem\u003eDn5\u003c/em\u003e and Tugela also hydrolysed the Laminarin-oligosaccharides with higher DP (LAM5 and LAM4) efficiently compared to non-concentrated enzymes.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Laminarin-oligosaccharides produced with non-concentrated \u0026beta;-1,3-glucanase extracted from wheat cultivars infested with RWASA2 for 3 days. \u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cimg 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\"\u003e\u003c/div\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Laminarin-oligosaccharides produced with concentrated \u0026beta;-1,3-glucanase extracted from wheat cultivars infested with RWASA2 for 3 days. \u0026nbsp;\u003c/div\u003e\n \u003cp\u003e\u003cimg 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x0NDgzUWOyUHt/C+pwXgJGyhzRU9QOdIftGx6xtacb+26rmka6j1aLZjLkutEEIIIYTIPRK1QgghhBAi90jUCiGEEEKI3NNyPrVCCCGEEKJAM8lAdRQTIoBz5NntX0pjIm+85B/14NoX/FML7tZWPZOWPfnXdE60Au/d/wvqKCaEEEIIIUQjIVErhBBCCCFyj0StEEIIIYTIPRK1/QSf18Nfs1LI+nxmX8C6evqVGZaL222hJwwfPrxLPa36BZzestugz7rrbvxlGitw3qdv9ukWqsHyYflNm3ekOaJWXrzHLPf45mfTWAHipFv46mWr0pzqnDvjpm6Vb2T2PehS96LdZ5WEpdc/kuZ2ZfW9m0vKEi8H9VCm0dkpaePG6PyIYTspZ2H8hKvSnAIsH+ZbqFZvM/HbB7d4f1ALF37kB2lOJ6RVyg+Xt0C91Xh62w5fNiZu05rlv0pzRH8hUdtP8AlEnLEt8OlRPnsYpuXp06Nhuwlz5szxgjTrU6yV4FOafH4wrKvc5zNFeU6eXHrTAwTtd69a6zu+EW694cMVhe09aze7D09b5h64e7ov/+0FE9xbD71UwrYbIFhjELSvTcTchReMc3/5w2wf7li1sSahiqC9YtHaNJZ/Nmx61q285XT39z/OLoaJ794/zS0FgTb6mCuL5ZkSzxK2lJ08dVkaa1wQntVg+0Yl27kq2d6OZLsJ6x9/pkTYbn/qz35q+RaG7r2bT292EI+fO+177gtXfcD96JHP+fDU1mdLhOt3Zv/ETy0fwnyEKXzz1nOLZQivfcsQn14Oljv36G+msU6sTR+de1KxrqXzbpew7WckakVd4DvaCHW+f18rZpnOk5hvRLCuPp6IhRCEKIIWYWocMmJv98HTRrhvzs8WU5d8/U6f37b3rj5+4vFvdPu07eZu+ol+lKux7PpHMgUtfO3y1W5osh8/cfaoNMW5eXOPded/sT2NZUN9zSRoTYyOPHhvP63GV5P9Nu7IfYvlmRJfcu3DPh5y1idvdsOSfdyoYEWuRdDCmrVbSrYbFl12kmu/47GiJdbKtCq/f2ire9Nhw0oE6JmzT3APr9ngRSdhxbL73IlnjE5zk9+zZN7yYcOvnnCv2GtX94rBhd+7Wrjpe3dlClq47oqVvk2HjX9DmlJoE8JW9B8StQ3G3Llz/Sv5kDgtdmUgn2lImB/XFxO7AFQrX47Jkye79vb2orWWumJ3hdC1YNu2bW7YsGFpTPQExCvW1cu/fmKaUspb3zw4nSswZK9d3MrV2db0axaf5i656Ng0JrrD5y66zd1+8+k+ZPGOI0qvqX1Si9pdZV6nY8U9Y8oIb9VFEDcD27Y/3y3hefnc49xPl52Wxsozd94qb8n80vlHpSmNx6wLb/OWV0I1Zpwzyi2vst2btu5IzovaxVizcdwHDnfnf+uUNNYVhGo1q+ufnnzevXKv7l1bP/zabd6yizU2izccvE86V2DX3V/qnk6OlQlp0fdI1OYMBO2oUaOKr+x5fT9z5sw0twDiEXcAe50/depUn5bF+PHj3dixY4tlqY+AUO4uZnHdvn27n8KkSZOKdS9ZssTHTfSuXLnST0PR25P1tjLnffom9/lZY70VNounnn4hnSuwZetz6Vx1sOhiAT73zE4Lo8jmN2unu8MrWCA3Ri4csc9tDFbdbyairplYdfcmPw19ZBGktYK1E2vlJ87qfLOD5fJTX2x37ddMSVMak/Xrptdsoc5i8bUP+wcCcy9Yccd6H0J/2kr+xq3AXT/5ZUXL6/xZN7ixEw4s5v/q3se9y0J3fGARypUsu398ovT3dccfC24iov+QqM0Zixcv9q/5TUAOHTrUC1gDUYivLu4ABvNYRLM6YM2fP98Hg/rqaT0N22a+siZ6TdyGghqBLmFbG5VEJy4EY44Y7s762HVpSsFnFpeEalAO39vPz17hBbPoHe896U3uZ4kYw0XBOGfGzelc67BxU0HYmy/t+vume0FaTdgiXBHA+MxO+8CIEr/RcScvchdfMK6pfUkR8wu+t9a7IBj4Jo89cnjRl3bJwgneD7dVhS1iFHcDXvfH4FuLYMViesyUw9LU5IE/EbRYas3/Ff/cb8y4tsc+sEec8GbfhrCjGUJa9C8StTljxYoVrq2tLY0VGDKk8xXLpk2bvAtAaP0kIBi3bOnaqxMRC/jChmWppx6EbTNwO4Dly5e79evX+3kwgb5w4cI0RZQDtwNE57VLyluocCfAJ9ZGMsBnthaRitXXOpfhqpDVCU3UDhZc3BLef8ay4ugHn55+RJrbOuBK8Ni66Wksud4TIYogvfLqdWlKNpQzIQyMoACI4eH7vMy/rm9WELSTEjE/J9lPoaUXITs/sOTT2Q4fWyy6rQYiFDH6vo8flelu8KFZ7/KiFbcB/GFNtH7zJ+eWuDCwLJbcO294KE3pHvjS4pZAZzGz/GaJbNG3SNQ2IVhyzfoZhtB6a5iYRSxbuZ5aaq3j16BBg/y0J2SJYNEV67zF6AQmWgH/2lCEImxNoDKP+0FbN6xa533sCHf7nZ0PHqJnIGxt5APCoFe+1KfbtFUZMnjndK42cD3ASon1FjGMO4K5MtjoB8wjBvOOCVqssLUI91b0sTVBi5jEz7YSuA0gWn9zX3lr9u577pLO9QyErVl+CUYllwVRXyRqG4wsURdaTfF/ja2ooQUWKy4CtRZ4/b9gwQLvnxtaTLHU9gRcI3B9MOtvNRDTsUsE29LTjmqtBC4HJlYtAKMdIF7hgMO+1mXcWtwPDjuk1NJvUD4eGWH7k6U+uaL74HbwuhEF66Jx3c2P+g5g1mGsFcgSm1u2Pe+trVm8c8JV7qwZN6WxAjaUFWD1NQsuYXEi/oD5csOE5QWs0CZo423BxQAfWhsJwcC9o22v1hFPjERggjYccQBsvNi4g5bFmZIfugoAPrE9FaC0Jx4LFz9fRkQQ/YdEbYMxePBgLyrN6mnC02CEAeJhfthRDGssy4dDa9loCbGANPFp7gBAx7GegB8s7Qr9c6uBRZmOYwbtZFtmzWr8AdTzwJFvG+6+POe2NFYYtxZ3BIbqyuK0Uw70Lg3huLRYfuVX2zsOeuvgRHA8W/SppZMYw3l94dON21u/L8AfNhxLFnGGT+1nPpHtinHapLd4X9LQT/QDZ1/bxa+22UD4z0z2CyMlZIlz3BDoNHbmJzv9shHBWK2b2RUjBAstIxHgBxsLWsCVgE5jNlYtsAxDeuFXi3BFbIY+r+TjExv63XaHV795L1+/CWWm8bBiou+RqG0w6ACGXykjHCBEscyGna3Ix7Jq+bgKkB+6DOBCgMAkn2CjJWR91MBGJLCyY8aM8WKzmrXXyltAjLLeWq20gABmXVaHtVPj1tYHhucKfWrv+MV6d/+aj6e5hY5mWGcNrL/2wQVbBkGr0Q96B9bY0KeWDzF8/4oJbkIgWEgPO5I1IwzRhSA1dwH7sIL5iiLmSDcQdPbBBVuGzlHU02xgeTUr9qIlD/opHb/C0Q0IJvAZTYFhzCwdEYyfbatgfq+h/6oFE5X4zIKlY9XFr9YssfjT0lGsXL4fk/Zd2WPSZoGQDn1q7eMQlYYVE/Vnp0SIdKTzTQ/CqRk3FyspnatCFwLRMzhH7FW+yB8v+ceefa5ZVOafWnC3tuqZtOzJv6ZzohVAgDeTLpKlNmfgVhD7nGIlZSxaIYQQQohWRaI2Z5jPqr2yJ+B+kDWygRBCCCFEqyBRm0NwM+B1gQUJWiGEEEK0OhK1QgghhBAi96ijmBABnCN/a6FexE2HLu8+4UXqKNYynP/7v6RzohW46PAvqaOYEEIIIYQQjYRErRBCCCGEyD0StUIIIYQQIvfIp7af4BOwfDGrEv31NS32A18Sy/rCWDX41G74aduQ3uxb9s+UKVMG/AMSefepPXrCVW7FHY/5eT6l+bt10/18Oe66d7N72zFXpjHnfrhwgntvnr+b3wC/Zv+4xyz3+2S/x59yJd34yJQR7rIqX8Y6e8ZN7luL1qYx5/72h4E7L/vKp5YvZPHFMGNxcv5lfRrWiMuHXySrN33lU8sXwDbc1/X8KAfbzNfFbBm+PDYp+NxwyLgj93XLl52WxnpGnnxq8Qc1jjzz7e6Q95V+4vaeH65xd8z/eRpzbtqPz3a7DCp8MQzaL7nVPXD9/X5+1z13dR9Zdrafr8Zz23e4Be+5zH36rs+mKc59a8JlbscTnZ8YD3n/gilu8P57pbHGQj61okcgVjlxLPB52HHjxpWk5enzsGG7CYyViyDcuHFjWqJ2ahH8ojoI2g2PP+NFOeGL5x/lXnPQpWluV36U3BwRtL9IhAHlmb4vuVkidEXPCIVryGtGXOouumCcF6YExOpXLluV5nbl6Pde5X525/pieUQwdTQTiDP7VO7fk/OP6eTk/LNPwcZs3Pxsl/LEy5VvRBC03QVBG4Lo55O4YViSPAzA/K8c66etACISIYuwJCBeEbEGgpU0y6csQtSg7ONrNxbz9xkx1NdZDRO0MQhiq8sCQvmt7z6gYQVtMyJRK+oCY+Ui1PniWXegvARt70GIYqH97mUnpSnOW1yH7fMyL16zuODC27zQOjy1dDH9yAdGuCXXPuzjonbYx+UELXkbNj3rPnl253n+wysmuG9fvS6NlWLH8qc/npKmOG/VpY5meuD4bHL+XZycf2ZpZTqtwvn31ctXe0tkWJ54Hs5XBHxPBO2ZM27yb1yqgeUWYVur9Tfv/Pq2R71VNLTMIlofuvEBP4/wxAKLhdSgLCKTZQHBO3rqkX4eDp58qK9z2yNb05SuIISzBG0WJrDHnXe0n4r+QaK2wZg7d26Xz+DGaVg2sYpaIJ9pSJgf1xdDfnfKl2Py5Mmuvb29aK2lLtwVwrqJG5RbsGBB0dIreo8JVGNo265u1d2b0lgnWL0QSYeNGJKmFEA8VXstLrpywUW3uV/cfLoPMVueeN6NTcRXyEFvHez3P8chZs26LV7IxAIFi218fPNKufPv8uTcI2RB+k97+Wp9oJiVCPhVt5zuQ60ghBd8b61bFDyoZmHCt5LbRrPx+qP285bQcmz/9XY/jS2kWFNZFlje5msFIYwLw/FfODFNyQZRHYtm0T9I1OYMe1WP/y1icMOGDW7mzJlpbgHEIyKRfMLUqVN9Whbjx493Y8eOLZalPgJCubuY+8T27YUfFMD/1urGj5e4id6hQ4f6dFE/YpG0cVO2j9eTT/3ZT1/1ype6f9x9VjGUs+qKyvxu7fSygnPzlh2JQO304wMTrHYcQij/jiOGe/cErL8Wmont6XYPSs6/FyXnnQWEXK1Qtv2Ox9wnzmp8t63166Z32/cX62s1Ecz1jvCdff5RaUprEovIF55+3g09eJi3yuIzaoFy5Wi/5Kd+mUquAgjh0Ce3HPcuvttbhbsrmkXvkajNGYsXL/av+U1AIgxDKydiFF/d8NO5zA8bNqzESmrMnz/fB4P6KFsvwrZZx7RQ9Ir6gKDCWnP2J29OUwqvva3TWDneefKiog+u+dRK2NaXjZvL30i3bX8+nSvFOoiFPrXNJmxhXHL+4R8b+tRWE7YIOQQwZXFXaMZX7uMnXOW3rZoQvuamR1vOShuDHywuAbGI3HjvBrf1oc1F/1bzqY2FLW4CCF7KHz6lPq5wuD7ISjswSNTmjBUrVri2trY0VmDIkM5XeJs2bfIuAOErfwLW1y1btqSlOkHEAr6tYVnqqQdh24xt27alc6KeMNIBHcXM6orbAT6ylfjpNZ0+ZwhjfGzxtRX1I7bShgwetHM6VwruCqEPLi4hiJdKncvySHtw/iHg8LHF17YSiFgTwrBvhc6QeQRRvz65jufX4Aa08Op1buqpB6Wx1sQ6aCEiQ2ssIjf0Z8WnFkssVtQQ0k34fn/aopLOZj3BfHZlpR0YJGqbECy59so/DKH11jAxi1i2cj211OIaAYMGDfJT0f8gbM3yihDC/WDvvcqLqtjKNWRwtsgSPWfvIbt2sdaamwjuH1lkCWE6/TUbvT3/cD0o55ucVxYtedBvEx3LCDb6wbADL3Vz53U+1LDNWX7JrQoiEiFr/rS7Du5qwd9l0C7pXDZYczff3zuDDtZhxLMYGCRqG4wsy2ZoNcX/NbaihhZYrLgI1Fqwjlr454bjw2Kp7Qm4RuD6YNZf0b9gnQ17x3PTw/0g66Zn/p9xb/ot255vSvE0kAzZc+cubiDrHii8rch6dT7q0DY/nFcMVnjqagbstXo8HBfn3/Ay5987J1zlzppxUxorYL65zQTjzIbDdZlfLePUzjin03q/Nj2Huuur2wxgTc0afsvGiR38xr28O0HMc9ufczu/ahdvzcWqG4908PyTz6VzPYdhwvY+oPRtqug/JGobjMGDB3tRaVZPE54GIwwQD/PDjmJYY1k+HFrLRkuIfWpNfIbuAHQc6wn48tKu0D9X9C+8sv7yV+9MY87715JWrgMTrgbhhxcQuJ/+Yrs7bdJb0hRRD/zQavg7B4LsfWcs8/s/C/v4RVieeaxyuf4wRgSuBuGHFBC4n6pw/pFOp6hQCH/g7Gub1q+2GjwAMKRZK/LaI1/nBWzoKsC4tOZXS2cvrKU/mr44zS0IYYQuy9LZi/xbZt+Y5ib3wUTg4gvbW79a2oWoFgODRG2DQQcwOlcxwgFCFMts2NmKfCyrlo+rAPmhywAuBAhM8gk2WkLWF8RsRAIrO2bMGO++UM3aa+UtIKxZr6y0A8ety04r8am1NOMr81aVfIzhk+eM8l8Qs/II3Nx/UaxBYXQEOn/ZSAZ0/Ap9ZkkLO+hRHpcFK8+ydBhrJrA68gUxG/kAgRt+UQzxSrq5FpBuH1ywZcYeObzsEGB5AjeD7oz8AJu27vBD9rUiiFJ8YBnxwEY2wEIafhHsvZdO9u4Glm/DcdnoBeTzwQXLx582/PJXOWtwJcyf999f/u9+KvoffSa3CcBKunDhwgH/xGwzwDmCP6rIKRohrk/oq8/kNjItuMmePH0mV/QeBH0z6SJZanMGbgXxxxGwkjIWrRBCCCFEqyJRmzPMZzV89Y/7QdbIBkIIIYQQrYJEbQ7BzYDXBRYkaIUQQgjR6kjUCiGEEEKI3KOOYkIEcI7seFIdxfLKS/4hnRF15R/VUaxl+MH2v6ZzohV4/5u+oI5iQgghhBBCNBIStUIIIYQQIvdI1AohhBBCiNwjn9qcwOdr29vb01hXxo0b55YvX57G+o5m/9BDnn1qd33VLPedBRPciSd0fhGMtJif3ni6O2RE9qdzr7vhEfehacvSmHMP3jPdteXoE6SN4FP7z3vMcr9bN93tk+63Y957lVtxx2N+PuYHV0xwE8p8we21Iy51GzcVvqZ1xpQRbt4AfjmrXj61+x50qf/cb0j4FbGQrLIGXxYbefDebu68Vf7TuiF8OvanwZf0ekpf+dTy9bAN902v+mnf8ROucu3pecNnltcn51RImM82L6/DNkNefGp//+AW96Up30tjzr3xsGFuxoJT0phzf9i2w33iXd9MY5189Sfnuj0Gl36J7Z7lv3Irb3yoZPlKWN3ff/hzaUqBuE1nzTnJHTL+DWmsMZFPrRgQEKyceIQNGzb4ND59a2n9IWhF43LypKvSuU42pZ8XRZgi1C2UE7T3rN3sBa2VRyC/5ZBLi/WI6iBoY2750Wnuf/8wuySMTUQIoZygRQi/44jhxfI/u3O9O2fGTWlufkGkIkj//sfZxZAlaOGxRMSF5QiIu2kfGOEFLWzeusPHwzL1ELR9BYK2Foann7PuSLaHMHyfl3kRa5yZnguWD2F+s2Pi8bOLPuCFJeHprc+6udN+kJZw7rk//tlPLd9ClqC9fOa1aaw65cSytQkha+v68WW3+/pF/yFRK0TOwbq6cdMzaayTBx7c5vZJRECtltZLvnan++BpI4rlsfiy/I23POrjojzLrn8kU9BmQVkst4jdLO66d7PPn37WyDTFuS9++ih3xaK1aSyfrE62C0yQdhessnB5YLFeccd6N+rQtjTWuCxNjnmtgpayiP/Q8jr/K8d6q+zG5AGTsOB7a92sTxyR5jo/b/mtwGMPbfWW2Ve/ZUia4twZXzrB/XLNBi86wcpUAhHcHUH7k+/dlSlo4YaFK/36QsssbULYiv5DorbJ4PX50qVL01iBOA1XBtII9tld3AoM5i0/XjaGvLBstfKivmBFxbo6/xsnpSmdbH3ieTe07WVprDrXLDnNffXigXvFnWcuuOg2d8fNp/tQjVPOWOYuvGBcGuvK4Ynowzpr7gvNwrbtz3tLa09ArOFm8KXzj0pTCiD+Bg/aOY01LrMuvM2tuuV0H6qx8u5N3p0gBFcFLLJMbb6nDwfNwLs+cHhVV4E/bn+ui1U2BMsq1l0sqm+fcGCaWpmlX7/Nuy9gjc1ivxH7pHMFdtn9pe4PW3cUhbboeyRqWwwELZjbApg7AyBoZ86cWcwnTJo0qUT0GqtXr/Z5LG9lp02b5tNE/3Dep252n581LtOlYNXqjd6Ci1+tBay6tfKNy1e5xxPR8NGzRqUpohy/XTvdi9FqXHJZwdp43tnd26fVhHAeWJWINXjR7rOKwayv1fjq5au9IA5dFczy+4Gzry3Whx9uI4I/bK0idOOmHW7MqKHexQDrLsHcEcoxJdkHuGFU89NtZtbc+ku3x167FoXsL+/e4AM+oxYQsgZW3q/ecm4aq40s94UQhHSIuUCI/kOitoXYuHGj72w2f/78NMWVzAOCFl/dkCVLlvjOYTGDBg3yQnbo0KFpinOjR49O50Rfg+hEtJYTneRhqTVfWjqIYdWtJmzxrUUAf352uxfMon58++p13RKn+NaaW0N3hXCjgVgD831df990b32tRdjyuj220mL5he9ddlKxTnxPG1XYdoeZyX4ZfWhb0WeW7coStiZ8sVifF7irtBr4rf582X3+db+BhfSNhw4r+rdiXcXnNRS29WT08W/2bQjrv+KzN6Rzor+QqG0h1q4t+OSFIhSGDSv4HSF6YdSoUSXuBGaNjbF6WC4sa2mi78DtANF53dIpaUpXHrh7uncpMLDm4jO7+EcPpinZUM6EMNberE5oovvgK8toBicdu1+aUh3rZIZbA+KWOvIKHbjo/GVgVbw4EfhXJkK/EviYQtyhjDhCNrSAXv6VY73AMytuXsHqGm4v/rVsl+0LY/7c47zoZTSFYQde2iW/FbCOXhM/dlSJjy1Cdspn3pXGkt+18W/wPq9YdPsC6jfhbJbhUGSL/kGiVnQhdCcIQ4yJWUQx1lzKMBV9j3XeYnQCcy0ALLGVROiQvcq/OsvivI8f4X5+Z/ZwVKJ73L1uixvatluPfGVxbWC0BOpoJoYMru4Pi9tC7GNajmZ5/d6WcZ1W8kdmuxHC+OO2EiZoEZP42VajkutAPUDYmmWYYPT1ekUnErVNhlldjdBiOmLECD+NrahmhTXLq1l0q3HJJZf48XERsxMnTvRpW7Y01023UcHlwKypFoBhuLDOYslF6OJKELJl6w63997ZP7BvPfRS79IQsj19xSt6z52rNvphuqpRbiSF9Y93HeEiT+DzGlsSt2x73r9arwQjHBw5qvTtEpw14yb3zmgYK+v9P+iVL/XTPII/7e3JuRJjneKwQuNyEI90YO4drQIjEZigjceCxQUAS2ncQYv47oN2SWP1hfaEQ4oBVuFqIzCI+iJR22QwksGiRYvSWEF4GohWRCgjHhjhPMyZM8e7EITCl85l1BvT1tZW8hEGOo7hkysGHoblevsR+7ozP9o5XA2+tN+9aq07Z1q2791ppxzkXRrCcWmx/Mqvtj4gSmsZfsrGrg3HpaWDGa4LefarxZI4eWrnhz0QZ/jUfiYYmioLxNxhIzpfKxuTTnqTH8YqFMrjTl6U+w5TM84Z5bcr9DVmDFostbhaEJg/85M3p7kFFw2WaRW/Wiy0jETAOLWxoAXcEPbYa1e36Ms/SVMKopMhv2qx6PaEfd+8l6/ffGqZ4mN7wlT1M+lPJGqbDD7CQGcw83GNO27ZRxosH7DuDhlSuGnMmDHDC1vSwjJZXxCjLFg5fHHN6lurtVf0HVhs6Shm7gn2YQUbhxarLNZZA+uvfXDBlkHQavSD+oAo3bPM8FNYZrHQGvbBBdIJ5yfij7Q8w/iyCE4bqWD0MVcWvwwGCDPSQypZXlmO5RHKVufYI4eXjGObF7C8huIcP9mFV68rjn7AA1H4RTGbt/xJyT6o5StlzQJf/4LQf9WCiUpGNmDILktHBIcuAdXwY9Iekz0mbRYI6dCnlimiO/TzFX2PPpMr/H7BF9ZcCFoZ9oW9yhf5oxE+k9uM1OszuXmiBTfZk5fP5Ir6gABvJl0kS20LYR27wo8j2PizErRCCCGEyDMStS0EPrVYZPGZRdwS7EMLQgghhBB5RqK2xcAii4gNgxBCCCFE3pGoFUIIIYQQuUcdxYQI0DmSb55VJ78+4d9bsKfYP7doT7HLNv9vOidagXMO/GJT3fNkqRVCCCGEELlHolYIIYQQQuQeiVohhBBCCJF75FPbT/AJWb64VYlVq1a5kSP7/jOH7IeefmyBMW4ZEiyLnuxbPr9rXyEzBvJDEPKpzTcD5VP7yU/f7D9BHPLB00a4r1x0bBpz7mWDPpvOdc3LIq7zme1fSuf6n3r71N5172b34XOudb9Z2/mVrBfvUfo1sZDfrpvu9om+lnXMe69yP7vjsTRWyvevmFD83HBPqadPLZ8EHnXMlWks+a0PvqSWRVx+3JH7uuXLTvPzfHmML4hlEZbrKXnxqd3w0Bb39Q8vSmPOve7QYe7Mee9LYwXmn/ND95u7C/eXlw/e1X3uhnP8fBZfOGGeO/zEA9xRpx6WpnQlrM84/tyjSpbBR9UYdfKBbsKnjk5jjUmz+dRK1A4QZ555pv8Ygn22tj+ph6iN9yMfcWDMWwQq4+HWCm3pLzFfCxK1+WagRO17Jl/lRo0c6s49M/vB9cDDvuZOPeXAYj4C93OzxpYtT32Pb3rW3bfm4z6OwL3jF+uL8f6mnqIWQTvm2Cvd0LbdSkRtFgjdCy8Y5z5xdm2fakbowi0/6p2wg3qJWj71O+zAS4tC1gRrOWGblT/8oEvd8H1eVlawmtCtx6dy8yBqTdB+7NtT3LA3Fz5Diyjdfa/disIWAfrHrc8Whez9Kx51t8y/PVPYLrv4Vrfqmvu6CNQY1nHMmWPcAWP3S1M6+dMTO9wXjp9XUgeCsVqdA406igmRwYwZM9y0adO8WK8VrNfQKIJWiJ5y+53r3YgDs7/xft2Nv/QCNRSwVy6Y4K7+wX1prJR71m729V2zZEqa4rxVlzrIyzPnzrjJC9paoOw7jty3ZkG7LBF2WG7rIWjrySWXr/YWVBOoTIkvvvZhH49Zs3ZLSXlYdNlJrj3ZNgRyFgjaJQsn9FrQ5oXHH9nmLbMmaOGUzx/vraiIS0Qv86QZCFFEL+I2hLIIWiy5laDeP23b4fbeb1CaUsrt37/btykUsFMuPMnddd39aUz0BxK1DQYWT17Jh8RpiEEsihbIZxoS5sf1xZDfnfLlmDx5smtvb/cWaKAuLLth3eEnerdt2+aGDRuWxoTIJ5s27/DTV77iP/w0ZtsTz7kxR5ReU29982AvUm3ZkLX3bXH7tO3m2vYuvcnifnDIiPKvrBudxxNBdsWite4vf5jtra+VwJpL2U9PPyJNqc77z1hWtd6BYP7c47rlEjDjnFHdKn9mIv6HJefLxF66W+QJhGPsapBFKHrh5Xvu6jY8UPpgiMUX8VmN5//4Zz+ljix+c88G9+qD9kljBRDSlVweRP2RqM0Z5pvLK3teGfC6n9f+IYjHOXPm+HzC1KlTfVoW48ePd2PHji2WpT4CQrm7mMV1+/btfgrmqkCwT/Sa6F25cqWfhqK3J+sVYiB54KFtXoQecOil3q2AgPuAsXXrc4lALbWgmWB96ukX/DSE8ke+bbj75vxVxfoIeQefWARtLVx06Z3eSnt4xuv5LL562So/rdWqO5DgKoDV9byzan9DhVUX4RpbYrHcLvjeWjf7/KPSlNbl/uW/8tbWUHRiXQ2J47gd4Pea5U4Qg3WY+nldb4HlDay4++w/2LsoZOWL/kGiNmcsXrzYv+Y3AYn/KgLWQBSOGzfOuwMYzGMRDa2kxvz5830wqK+e1tOwbebDa6LXxG0oqBHoErYiT2CJxep6/93TvTWVQNyE7aYyr4xh+5NdRS1YBzGrj45lzSBsawGLLm4E3bHSfvvqdQ1ppQ1BgO60+yzvKjDtAyNqdhVABCNccUGIueamR1vOSpsFLgW4EJi7ARZaBGgoKikTdvKyZWrtyPXs9ue8cJ133wXFwPLhOrD6nnX5KWXzRd8jUZszVqxY4dra2tJYgSFDOl+xbNq0ybsAhNZPAoJxy5YtaalOrFMXvrBhWeqpB2HbDNwOgE5y69ev9/NgAn3hwoVpihCND76yCM/QXeCyr5/o/WJxL4ittCGDXpXtsoC7QuiDi08t1mCst83OdTc/6juR1WqlxVVhY/IQceKx1a1tAwkituOPs30AOn9VwzqAzUkEe1ansoWJmJ966kFprDVBnC46/1rfISt0N+C1Px3FzGqK2wFWWYNl6GhWK4hfhGoIbgsIV4N4aCmmfvJjC7HoOyRqmxAsuWb9DENovTVMzCKWrVxPLbXW8WvQoGxH+lrIEsFC5I3Qv3avvXbpYq2t5oebJYQRta3AHas2undEPsiVuGfdFi+C4yG/GhlcDzYkQrxcxy8wQUsHMPxsY1iWOg4b0bq/mSZoEZNZIwwgbM1qiihFXO42aBe/HGBZNdGLFfbGb97m3QdqZZc9XprOFYjjO+9eGhd9j0Rtg5El6kKrKf6vsRU1tMBixUWg1gKv/xcsWOD9c0OLKZbanoBrBK4PtQ7phZiOXSLYlp52VBNiIMDNgCG3QsxXFuvt4D138VbbEPxwIe4MBoce0uaH74rBpYG6mh1cD0YeWvo2qhLdFcH9zfgJV/nOXCHbnyp0OirH3HmrioK2nGvB2gcK51CWBbcVuO3qNUVBm+UT662zD3XeGxG0uB/g90p5E7sWcFfA2luuYxdil3WGPPeHPxdHTWDkA/xuQ6p1LhP1R6K2wRg8eLAXlWb1NOFpMMIA8TA/7CiGNZblw6G1bLSEWECa+DR3AKDjWE/AD5Z2hf651cCiHH7IgXayLbNmlR+EXYhGY9J73+p9YMPhtt51wrf9OLRw4vFv9FbWUPiePm1ZMT+G8hCWZx5Ra3nNCv60cNBbB/tpLWx4/JluieD+Zsqkt3ifWMafNaacfW1Zv1ostDO/2O7Hqa3kK7tl2/N+6K9WBEsrVlVe75fr5IXIbP9Op7sOvq3xMGDdgQ8zsE5zJWCKqGbcWhj3oVE+PxTSWIJDlwfR90jUNhh0AMOvlBEOEKJYZsPOVuRjWbV8XAXID10GcCFAYJJPsNESsj62YCMSWNkxY8Z4sVnN2mvlLSBGWW+tVlpAALMuq8PaqXFrRZ5AaDLuLELWRiqIP6zARxMQvpZPx68wnzTGszUoj8uClWdZ/HabHbNgZrkS4DvLxxhM+Br40+45aOc01nggTBGofFCBjmKEsUcO90N9GaQhZmHRkgf9NCxvIRTGm7bucEPbWtMCeO/ND/lp6D5gwUQlQ36FPrWWVitYZUNXBNwbsOTygQXqYxpaiRHLn7vxnJI25eGLYs2GvijWBGAlpXNV6EIgekazniOtwkB9UazZqfdncvNAPT+Tmyfy8plcUR8Q3810z5OlNmfgVhD7nGIlZSxaIYQQQohWRaI2Z5jPqr2yJ+B+kDWygRBCCCFEqyBRm0NwM+B1gQUJWiGEEEK0OhK1QgghhBAi96ijmBABOkfyzV//+OV0TtSTVrwi/qlFO4rN3aiOYq3Epw5WRzEhhBBCCCEaColaIYQQQgiReyRqhRBCCCFE7pFPbT/BJ2D5YlYl+utrWuwHviSW9YWxavCp3fDTtiG92bfsnylTpgz4ByTkU5tvGsGn9p/3mOV+t256yVexLrlslTv/i+1prMDYI/d1t/zotDTWldeOuNR/LQuqle1r6n1F8HWwD59zrfvN2ulpivNfCyvHb6P9Cce89yr3szseS2OlfP+KCW5ChU/M1kI9fWrPmnGT/1RuCJ/JvTz4qljMi3bv3B8XXzDOzTin/P2DsosXTqj4Wd1ayZNPLf6gxjvPPsq97f2HpbECYT5cfO8F6VyBr5w0zz2zrfDZW5hx3Tlutz3Lf6Xtux/9ofv9PRvSWIFwvZse3uK+NXWRn4ePLJzi2t7Us8/y9hfyqRU9ArHKiWOBz8OOGzeuJC1Pn4cN201grFwE4caNG9MStVOL4BciDyBos9i8ZYc7Y8oI979/mF0M1QTt8H1eVix7xKihXsQ1AwjaMcdemcY6+UuynXGACxNBl/XZXPZfXP4difgn9FbQ1puNm3Z4Yfr3P84uhkqCdt+DLi0p/6nkgWjuvFVpbinvnNAc50V3QZAiKBGqhJ9edpv7xffXpLkFsRbmH3LSgSUil+X3PXhYMZ+yc0+c5559olPkxjyz7Vk3afZJxWUIJmhZDkGLkCWdKXGErug/JGpFXWCsXIQ6XzzrDpSXoBV5Z9n1j5QVtPCzO9e7UYe2pbHKUBcW2nlzj01TnDvv7FFu/ePPeEGYZ86dcVOmoM2CsgjUTyTbXgvsNyy3A2nRLkd70q7DRtRmsVuabMeG5PiHllkE7pVXr0tjnVCW86LVePhnj3oLa2iZRZSuveF+P29CMsw/YcbRfsqyCFCWH/2+Q30aUPZlg3d1v7z9N2lKKbbMXq8flKaUsvKHd7tXHzKsaJllSvyh9l/5uOgfJGobjLlz53b5DG6chmUTq6gF8pmGhPlxfTHkd6d8OSZPnuza29uL1lrqwl0hrJu4QbkFCxYULb1C5JULLrrN3XHz6T5kgUjdc9DOaaw6Q9t262KdxHJ797r8Wn0e3/ysu2LRWm9RxfpaCcQ7ZT89/Yg0pTrvP2NZ1XoHgo3JdsOgV77UT6uBCwHW2WpQ7+Spy9z3LjspTWkd3vSO/bw1tBwIykr5uBiQX8nVIOaFP/3ZT8stg2j+4Dfel8bEQCFRmzPsVT3+t4jBDRs2uJkzZ6a5BRCPiERzDZg6dapPy2L8+PFu7NixxbLUR0Aodxdzn9i+fbufAv63Vjd+vMRN9A4dOtSnC5F3frt2ujv84L3TWClmXf3QOdd6ay4B94JKmC9tSN4tcoh0BG0tXHTpnd5KW26fxnz1ssKr+Vqtuv3J2ge2uWHJQ8rwAy/1vq+E7rgMIF5xP/jS+UelKQXO+uTN3oI7ssZ91MxgRcX9YOxHxqQpXcFCC+UsrVh3scTuvf/gNKWUzY9s85ZcXBgs3DD31jS3K6wP/9vQGiz6HonanLF48WL/mt8EJMIwtHIiRvHVDT+dy/ywYcNKrKTG/PnzfTCoj7L1ImybdUwLRa8Qzc4T25/30+/MO6noI4vVtZywNX/Qc2bc5KdAR7MsoduMYNHFjaA7VtpvX72uIa20sGXb896dYP1904s+sjyg1CJs8a1FDCOKw05g+NdSR6XOY60CvrH4wiI4seBmgehdMuta76JQztKK/yt+t+U6du148jkverHwWrjn2vu6CFvWheBlfdTXHWuw6D0StTljxYoVrq2t1DdvyJDOi3DTpk3eBSB85U/A+rplS9dXl4hYwLc1LEs99SBsm7Ft27Z0TojmB5GKkA2tjvjLIlLL+chSntfvZtkFRkBoBa67+VHvflGrlZZ9yL488dhsQTPQIDwRskMDdxJcBvCzNdeEcjy2riCEsdJi4aW8WW7br5mSlmptPnntOV5gYqVFTCIqQ4gjevFvDX1sQ1gOUWx+t1mQx3pC6DSGsA0x1wYri+gW/YdEbROCJdde+YchtN4aJmYRy1aup5ZaXCNg0KDs1ztCiAJZvfljzKpLsI5iQ/as3S83r9yxaqN7xxG1+/Xfs25Lpg9yI1Orf62BlRZrLa4M19xUeI0eujMA/rWtOhICYKVFmG79deebQBO0WEzL+bsiaBG8iOPusvMelY8jrgdYd2OhLfoOidoGI8uyGVpN8X+NraihBRYrLgK1FqyjFv654fiwWGp7Aq4RuD6Y9VcIUXAjiIfj4hU7ZIkb8rDOWhkwa+RBb83292smcD0YWeNIEdBdEdzfIDQZpzZk+1OFTkeh9dbAtQC3gxhcGMAsv2EAxqn96bLGG/mhL2DoriwLKALSwEfWBG2WBdbcBBC0tXTwYn3hkGHw/B/+7IU0MIZt7IpgnctE/yFR22AMHjzYi0qzeprwNBhhgHiYH3YUwxrL8uHQWjZaQuxTa+IzdAeg41hPwJeXdoX+uUII5yae9Ca3IhFqDDllvPM9i/y4tVnWRdKwPF56eeEaBzqZlSvfTJiQ74543/D4M90Swf3NaZPe4j+8sDpwNRl9zJW+k1cWJx+3nxew4bi0iOLYr7aVeeOY13kBG4pMBKX51SJY8ZHFh7acS0E1C27MiBMO8J3RzOrKFL9Z65x2wDFv9q4I4bi0P/7ijfKr7WckahsMOoDRuYoRDhCiWGbDzlbkY1m1fFwFyA9dBnAhQGCST7DRErK+IGYjEljZMWPGePeFatZeK28BYc16ZaUVohR8Qxnq65QzlhV9ZLEszgsG3yctFL2MphD61MblmxWzYGaJd6zVfHUstGADFuzuDJfW3yBEsaIiZM1dAEEbdvIijTFnAest1lf8Zq38ijvWe/9aUcD8VhGZWFsJj927oehCYGPNhvkWEMI2EgIiNM43a2tsDcYf1z7QQDmm+NRa5zSm9sEFq4uPO1Ty0xX1R5/JbQKwki5cuHDAPzHbDDTrOdIqNMJncpuRVrwi6vmZ3DyRp8/kit6D+G6me54stTkDt4L44whYSRmLVgghhBCiVZGozRnmsxq++sf9IGtkAyGEEEKIVkGiNofgZsDrAgsStEIIIYRodSRqhRBCCCFE7lFHMSECdI7km/96Wh3F+oIXtWCnqRe3qMln3mZ1FGslPnqgOooJIYQQQgjRUEjUCiGEEEKI3CNRK4QQQgghco9EbU7g87XhMF5x6OnnbbsLH3qIx8kVotVZc+9m95JXzioG4jFh/sc+dVOaWp7jJ11VLP/GQy5NU/PNcROvcv/6ilnFEO+nH9/wSEl+/PWwmLj8R2dW368DhX0djMBnb2uFz+uyzMZoX7xzwlXF+vY9qDnOj+6w8aEt3h/UwoJzfpjmdEKa5X/phM6vgxk/vvjWkjqq8cwTO0rK//zqzs/0xpBPedG/SNTmhOXLlxeH8NqwYYNP49O3lka+EKL/QXi94/gr3c9uPN39v6dm+ynxULAhSmd/dpzPJ1y5aK372uWd3/aPQfRufPyZYvmh+7zMi9w8g6CF/356tg9XXzHBvf24zv3E9NQzlrlfr51ezH/9iEvLClsEbViesDDZr40mbBGjCE8+jcvnbwkLvrfWzZ1X/viH8HndGATt+uT8sPq+dP5RLSVsEbTf+PAi99FvT3HfuO8CH/609dkSYcs8aZb/rjPHlAhbBO3qa+4r5lNXJWHLOr94/Dx32oUn+fIX3HiOu+mbt7kHVhQ+uRtSqR7Rt0jUCiFEL/jGgtXuqCP3dYcdvLePMyW+7PqHfRzxtXHTs+7jZ43ycVj0rQnuu99fl8a6gui94hsnpTHnvn7xse62Ox6rarlsZH6WtP9THz8ijTn3nhP2d+9I9tOPrivsp4u/dqebOmWE22fv3Xyc/KFtu7nrb+4qGmD13ZtKygNCGGHbSHz18tVuXLKdM87pPP6LF05wV15d/vgbWHSHJfsgBMtte7Ivv3dZ5/kx8d37u+HJg8/S6x9JU5qbxx/Z5l576DA39M1D0hTn3vf5491v797graMIUOZJM946dj/38r128yKUMghaBKpBXSNPPrCs9bX9O6t8PvXAy/bc1R137lHu3psf8nGgbgnagUWitsnAFWHp0qVprECcFroy2Gd3cSswmLf8eNkY8sKy1coL0Wx8/eLj3I1LTktjXdn2xPNe5IYc+JbBXujmWaR2FyypJvyzuGnpae4bc45LY9WhbHfKDxQr7ljvjhw1NI0VQIQ+tm56GssGgYpFNxSvISOjfTm0bVe3KhH6rcDbTz3MTZv3vjRWnlD0wssTIbrhgc43KHvvNyidK7DboF3c79c9nsZKQSQPe2vpPo/b8ZP5txcsvkkQA4NEbYthvrfmtgDmzgAI2pkzZxbzCZMmTSoRvcbq1at9Hstb2WnTpvk0IVoVLLNYVT86baSPb966w+2z965+3jDr4pNP/dlPY06fMsKd8dFr0xjuCDd7q2Volcw7CHqstyMPbUtTSrn0slVe+E8/u9PCWY3vL33QW38biQ3JNhw2Yoh3DzAf2Fp8aidPXeZW3nJ6GutK7GO7cVNr+2/ev/xX7uWDd/UWVCP2af1TFH/+j6XX37Pbn0vnSrF6EMFYYi3EVt3P3nBOFyEt+heJ2hZi48aNrr293c2fPz9NcSXzgKDFVzdkyZIlbuHChWmsk0GDBnkhO3RopxVi9OjR6ZwQrQUijU5dUz6yzItSE6CPby4vNp548vl0rhSsv28/Ynixoxj88p7Klr28ccyERV6A4mYQgm8tnb4+86V29+XPjktTq4MIRiR/Y86xaUrjgF9s+zVTSnxqKwlbfGanfWBEF2sskIZLwlmfvDlNKVh1cUloVXjtjzuBuRsgLBG4yy6+1ceBMlhbAeGL+8IPP3+jjwMuC9RRCXxqzQfXfGordRYT/Y9EbQuxdm3B1ywUoTBs2DA/RfTCqFGjStwJzBobY/WwXFjW0oRoJRCx1rELbMSC2Eobsuerdk7nSkHI7r3XrsX6jhg51Kc1i7vCfgdf6q2wuBzE4KJgHb/uXL2x2MGsEghaRDA+tY1ozcaHdmjQLiywCNvY2goIVDqBXT63vGsFrguUMcsvbgeI4FYEsXrV+dd6/9bQSorVlI5iZlXF7QCfWAO3AXxsLR+fWeqoROhWgDDGJxdhKxoHiVrRhdCdIAwxJmYRxVhzKcNUiFYH1wPzmUWcxtZaE6eveuVL/TQE9wVcDcKOZczjl3vDLdmdpvIEghYQrdWgYxnW10qYoP35Tad3sfo2CoMHlT68DMo47sZVSx70LgsmWG30g+EHXloyYgLC1iy/CGDcDzjXWgkTtIhL/FtjELZmWX3Pp4727gf4zRoIW8tnHvcD/G7LsfPupcdtlz3KH0cxMEjUNhlmdTVCi+mIEYUn+diKalZYs7yaRbcal1xyiRs3bpwXsxMnTvRpW7Zs8VMhWgWG2orHnQ19ZQfvubP3sQ2578FtflrOqoggbkZwK4BH7+3qSoHYRaCGlHPPMBi+ywRtpU5oAwkjH6xZW/q7uD09P0LrrfHTZacVxSrB/GrX3ze9OIICYpdREAwsvrgf4LvbKvDa3wStjUgQgvUVlwIDv1jcD/bZf7CPM7wXojgE94O4MxhglcWdYfOj29OUAs/94c8+XTQOErVNBiMZLFq0KI0VhKeBaEWEMuKBEc7DnDlzvAtBKHzpXJb1wYW2tja3fv36NFboOIZPrhCtxCnvfYsfgiscl5ZOXuZXa0NThcIXv1vGrc3CrI1heca0RRifcEzXm3deQLTiQ5slaOFD7z/IC9TQxYJxaMv51SKAGb6LcWobVdDCZz5xhPvUF9tLRCjW1964CyCUv/zVO9OY8/61pGX54DYjiFFe++MOkCVoAZ9ZXAoM/GvDYcBee8gwP1qBwbi1CNRy9THOLSLaOo0xJX7YiQf4uGgMdurIeq/cpPCqvBk2F8GJRZYOXSNHFnpYh7CdBu4AiFSmZk1FpNJhDBitYMWKFW727NnFfBsBwUAI28cdyKPTmIlZxG7ob8u8uSNYfXmiWc6RVuW/nv5yOte/IGj54IKBoKWzV4h1+II4nzzGrg1fn4fl4VeJGBwof9EXdf6k9Aj2Dx9ayAKha7619kEFA0Frox9YHYhY9oP55WZhZXrDi+to8sGSivuAgaANfWaxvOJ3y1BfMYhhRDCW2tCyy2gKuCkAghYLbz2Yt/l/07nGhQ8rWKevGISuCVessX/aVhChCNp4GLCwHgQt7goGluA1191fkmYffTDwwc1ye7BydCYLR2NoRLBoN9M9T6JW+P2SVxFab3SO5JuBErXNTm9FbR6pp6jNE3kQtaJ+NJuolftBC2Edu8KPI9j4sxK0QgghhMgzErUtBD615o6AuCXYhxaEEEIIIfKMRG2LgUUWERsGIYQQQoi8I1ErhBBCCCFyjzqKCRGgc0SIrjzzZPUPJTQbL/2HFuwdl3CZOoq1FJ8YoY5iQgghhBBCNBQStUIIIYQQIvdI1AohhBBCiNwjn9p+gk/IjhpV+DJOOcp9IazesB96+rEFxrhlSLAserJv4y+SwUB+CEI+tUJ0pV4+tQce9jW3KfoK2JULJrh3H/9GP//JT9/svnfVWj8PYV45wjrHHDHcLVtcny9r9YdP7bIbHnHvD76gdsaUEe6bc0q/RHfMxKvcz+54zM/zueXflPnMcL3Ik08t/qDGMecc5Y58f+nXva6dc6tbc+19acy5r669IJ0rUC0/ZuG5P3S/u6f0fhWvN2zTYScd6E6aeXQaa0zkUyt6BGKVE8cCn6fl87NhWn8I2noRtpswZ84cLwj5wEN3QNAi5sO69CEIIZoTxOctN3zY/Wn7l4ohFLR3/GJ9MZ1yp09b5q6/8Zc+P4uXD/qsO/Jtw4vL8Nlc6skDJmh/u3a6+8vTs324YtFad+7Mm9ISrjhv+cP2eZkXucK5C0+c5wUlQpRwy7zb3B3fX5PmOj//u3s3FPMRmCxjmKC1/LMXTikRpFn8aduz7pTZJxWXIZigfeaJHX75sE3UH7ZJ9D0StaIuzJgxwwv1M888M02pDtZryJOYF0L0jHvWbvbTQ0bs7acxCNrPzDwqjRXKfeC0EW7Jjx5IU0qZN3+Va2vbzX3lomPTFOcu+/qJJZbeRmb13Zu8ZXafvXdLU5z7/hUTvLCFxzc/6+c//fEjfBzmzTnWW23Ja2UeXPGoe2bbjhILKWLy7uvvT2POi9x3fmRMGnPeYsoyjz+0xQtQBCcC1djnzUO88C0nQlmG5YfsNyhNKeXOH9ztXnPIsJI2UX/YJtH3SNQ2GHy2llfyIXEaYhCrqAXymYaE+XF9MeR3p3w5Jk+e7Nrb24vWWurCXSGsO/xE77Zt29ywYcPSmBCimXnyyRe8CC3HfWs+XtXVIGTV6o3eShuCEMZimwdwM4hdDUK2P/VnPz384M6HAAQwLgjrHtyWprQmbxm7n7eElgMBCrEARXRu+mXnvovzdxu0i3ts3eNprJQX/lg4Hi/bc1c/jcEqvO9B+6SxArTz/OvOSWOiP5CozRnmm2uv7Hl9z6duQxCPuAPY6/ypU6f6tCzGjx/vxo4dWyxLfQSEcncxi+v27dv9FPC/tbrtE70meleuXOmnoejtyXqFEI3Pmns2+SkuAxawtlYCq+uokUPTWCm4Ghx2SJubMPmqYn3M55mrlz7o3nHkvn7+iSef9wI2BheErdueT2MCELGhZbacACX+7Pbn0lhnOSPMi0EMv2zwrt7FwAIuDAZW3LY3DvYuDln5on+QqM0Zixcv9q/5TUAOHTrUC1gDUYivLu4ABvNYREMrqTF//nwfDOqrp/U0bJv5yproNXEbCmoEuoStEM3H5vSVufm/3n/3dPeF2SvKClsEKpbdc84s38EWn9vpHzuixKc2r8L2q5et8q4FuBhAJeG6eWvBEikKvrUXvnueF5xYRuG5P5SK1RAEMOIWq+2SL96YpjrvlhB2GotB8CJczV/WfGZD4XrZ1EXujHmnlM0XfY9Ebc5YsWJF8kPflsYKDBkyJJ2jI8Ym7wIQWj8JCMYtW7akpTpBxAK+sGFZ6qkHYdsM3A5g+fLlbv369X4eTKAvXLgwTRFCNAuMSoCLgbH33ru6z80a6676QVchgTC9/c717tolU9KUbFg+9NGlPMuZ/25eQNCe/6V271NrPrZ7Dd7ZT7PYe6/sV+CtCK/3EZBYabGOIlp32eOlaW5XzHo79Zvvcy8fvFvRqvqz767yfrnlwCeX9YTgMxsKYeKhdZjOZ+SbO4ToeyRqmxAsuWb9DENovTVMzCKWrVxPLbXW8WvQoGxH+lrIEsFCiOZk8J67pHOdmKDF8orwrUS8fLXyjYgJ2ttvOt1NOGH/NNW5PV+1s7c8x2x4/JmKgrdVwUqLtXbLo9vdf+xeELWxmCSO36yBsDWrKvNYY2OXhUrE4jmOWztE/yFR22BkibrQaor/a2xFDS2wWHERqLXA6/8FCxZ4/9zQYoqltifgGoHrg1l/q4GYjl0i2JaedlQTQjQu+LzGw3Nte+K5Er9Rxpw1QVsNOomZn66xeXNBxLzqFf/hp40OQ3aZoA07hMGgVxYE0V33dlqdGfUAoYvgbWUYoSAcnsvAPQBMmCJwQxhjdpdXFPYdyzOKQghW1aFvyR6dg/LxyAi4OSCkIe6EBtU6l4n6I1HbYAwePNiLSrN6mvA0GGGAeJgfdhTDGsvy4dBaNlpCLCBNfJo7ANBxrCfgB0u7Qv/camBRDj/kQDvZllmzZqUpQohmgeG58IE1cBHApxafWDBf2FoELZz9kZG+I1kolE+atMh/gCEPFlsstAzZxTi1saAF3BAY8uvD516bpjh3zsybfUeyrPKtxP5jXucFbCgy8V0N/WpxJfjBrM59F+e/5uBh7qffut3PQ5wfc+i7D/Cd0cz6y5T6rXPaOz44yufjm2vgY8swYaL/0BfFBghEJ4IUv9IYBKIJVVwBGL0AP1OzpiL+wq+TmR9qaG1lW0PCr5WRZ1/tQuiGwpK6sARj7Q3rM+LyIfG+DddjxGnsh1C099dX1crRSOeIEI1Cvb4oFn8xjA8s4BOLhfWAQy9NU0uhsxi+uIjgY074tu9gFopWLMBGnr4o9rqDL810LwCErvnW6oti5cEX1kCQxsNnIVRDn9fYJzb8Qli8PIKZMWbjNISrgQ9tKIIRunRaM/RFsf5HorYJQATHolb0DIlaIbpSL1GbJ/rjM7mNSJ5Ereg9zSZq5X6QM7Bsxj6nWHWx5gohhBBCtCoStTnDfFaxKFrAZSBrZAMhhBBCiFZBojaH4GbA6wILErRCCCGEaHUkaoUQQgghRO5RRzEhAnSOCNGVF576cjrXOvzLP6QzLcZ3t6qjWCsx7QB1FBNCCCGEEKKhkKgVQgghhBC5R6JWCCGEEELkHonafsI+VVsp2Kdv+xrWFX8yt1ZYLm63hd7Atsfj7wohmpcTJ13lpn/qpjRW4O61m93Or5pVDNfe8Eiakw35Yfm4vkbkxXvMco9v7volMdItnDuj9u3gc7vdKd8srH9oi/cHtTDv7B+mOZ0suejWkjIxFxw/ryT/T+kncMtxX/ujFcuvuGpNST5x0b9I1PYTfPoVZ2wL06ZNc+PGjStJG8jPw3aXsN0ExspF2PLp3+4Sf/ZXCNHcfP3yVe7ndxY+/WpsSoTe+OOvdMtvPN09/+RsP/3gtGVe6GaBoCX/4Xum+/KE71y1tqGFLYI1C9LPmDLC/eUPs32AWoQqgvb8L7ansdYBQfvVDy1yn/jOFLfg/gt8+OPWZ0uELYJy5TX3FfNHn3ygF7EG8687ZFgx/93nHuU+e9y8ssKWdX7n/Gvdl246x5f/0IUnlZRnfdd/87ZifZQjLmHbv0jUirrAWLkIdb541h0oL0ErROuAeP3c7Ha3T9tuaUqBby5Y7d5+xL7u0BF7+zjTD502wv34uod9POauezb5/La9O+v57oIJXtg2Gsuuf6SsoEWYwjfnHuenwPwVi9ZmWnSN1424tCUFLWx8eJt7/aHD3PA3D0lTnDv1C8e7X9+9oSgyV193vxeexqRPH+3+uG2HF6eUYf4dpx6a5jo39rTD3O6Dd3UP3PabNKWUn357lRfGL99zVx8/cNx+JeV/t+5xL4wNyhGnHaL/kKhtMObOndvlNXycFrsykB+//g/zq73WJ7875csxefJk197eXrTWUlfsrhC6PVBuwYIFRUuvEKL5OWHiIveFWePc0LaXpSkFNm3e4UaPHJrGChx+SJu7/Rfr01gpl158nA954HMX3eZuv/l0H7J4x5H7pnOdDE1E/7oHtqWxUhDJw/Z5mbfqZi3b7CBAz7nsfWms+yA4saaaQK0F1ocwLgf5tEsMLBK1OcNe1a9atcqLwQ0bNriZM2emuQUQj4hEcw2YOnWqT8ti/PjxbuzYscWy1EdAKHcXc5/Yvn27n8KkSZOKdS9ZssTHTfQOHTrUpwshWgPcDhCzHzur69uZjZuecXvtuXMaKzBo0M7u8U3lrZUxP/zRg97a22j8Zu10d/jBBQt0Fhsefyad62Rjhe2e8O793S0/Oi2NCVj30195y6kJ1ZEnHuDdBQz8ayG07oZgwcV6O/RNg9OUyuBWQPlKQhYrLS4Oov+QqM0Zixcv9q/5TUAiDEMrJ2IUX93w07nMDxs2LLNz2Pz5830wqI+y9SJs28SJE/00FL1CiNYA31jcDi69+Ng0pZRK4hWXhWqYn265+huVE4/dzwtYc0OAVuz41RvowIX/LC4IBmKT1//Waes392zw1tly4KOLe0E50WtYBzX8ZUN3gxhENKK3knVX1B+J2pyxYsUK19bWlsYKDBnSeRFu2rTJuwCEr/wJWF+3bNmSluoEEQv4toZlqacehG0ztm3LfqUmhGhepn30Wu/zGvrAhsQ+tiHlljEQtAjmSvU3Kvsk7f3tuuneP9ZGPxh5aJt3PxDVQdBikUVghoKUjmDPbH+u2HHruDPHeDGKKI0hHStvLQKUdVid+NGWG3UBkU1HNtG/SNQ2IVhy7ZV/GELrrWFiFrFs5XpqqbUhyQYNGuSnQggBWGmxxDJagQ2/hVWVTl3MA24JW5943s8b27c/X1HsgglaRks46YT909R8gbC1kQ8IuBdgvd1zUKk7hijFBC0dwkI3AHMlCEUqHbuwxOKmEIKgpdPZF288J02pnXd+eJTvnBZigpbRD6pZfUX9kahtMLIsm6HVFP/X2IoaWmCx4iJQa8E6auGfu359Z2cMLLU9AdcIXB/M+iuEEMBIBjbslgV8Xxm9gHlo23tXt3J16ZCAjHAQdygLYfguE7Q2akLeuOvezV1GRqAjGFTyw2118Gk1QYtgzaLc8FxAngnaWjqdYfmNh+d67uk/p3MFsNqaoO1OJzRRPyRqG4zBgwd7UWlWTxOeBiMMEA/zw45iWGNZPhxay0ZLiH1qTXyG7gB0HOsJ+PLSrtA/VwghauXcaSO99dY+uIB1F0vuJz9+hI/HYKEln3Fq8ypowYRr6FP7/jOWuQsvGJfGRAwWWnxaeb2fJWixkOJOYJ3DwPxuD3rnG3ycMWax3NY6igIdz1hnKJTN7QFYF1Zb3BIkaAeOnTp439wiIOwaZXMRnQjS5cuXpymdIBBNqOIKwOgFCxcuLFpTbQQEg85YYT6wrSFYY61zGXmMREDHLYQuIxIY1IUlGGtvWJ8Rlw+J9224HiMrDdjmeBsGgkY6R4RoFF546svpXP3gi2JYZ8NhuRCyfIDBwEfWXAosDxGL3+ybD720bOcyK9Mb/uUf0pk6glV2zLFXeh9aXA4MxqN97UGXpjHnBe0nzu78jWdM2g+felBJmnHMe69yQ5P9GI5z2xu+u/V/07nGBYto/NrfQOjaa3+sq7ghGJZnbgtZIHRxW8Aqy+gFoVtCvByC1twesPqWA6HbqNDuZrrnSdQ2AY0iCJsBiVohutIXorbR6QtRmwfyIGpF/Wg2USv3g5yBhTf+OAJWXay5QgghhBCtikRtzjCfVSyKFnAZyBrZQAghhBCiVZCozSG4GfC6wIIErRBCCCFaHYlaIYQQQgiRe9RRTIgAnSNCdGXLtsJYsq3E7v9cOoJMq3DFFnUUayU+fpA6igkhhBBCCNFQSNQKIYQQQojcI1ErhBBCCCFyj3xqcwKfr21vb09jXRk3blzm18nqTbN/6EE+tUJ0pV4+tTfd9Et3zlnL0phz11z3YXfQQZ2fuJ11/s3u+99fm8acm3f5BHfccW9MY12J63v/+0e42Rcem8Z6Rz19ai+9bJWb9aXO3+/Znx3npqdfB/vozJvclYs6tzkkLFcO6t68dYf7xpz6fFEsTz61+IMax55zlBtzauHrXsY1c251d11zXxpz7mvrSr/sFea/fPCu7jPXd349LIsHVzzqrv5M5xfFzrlyihuafr0MNj60xc07fVEa65rfiMinVgwICFZOPMKGDYXPA/LpW0vrD0ErhBA9Zd26zV6AImQ3b/2SF6wnn/htt2VL4TOmCNqVK9f7PALlKI9wzcIE7aq7pheXQRBTTyNhgva/np7tw6Nrp/s46YAYtTwLp08Z4Ya27VaToA3Fcivx5XfP80IWoUq4ed5t7var16S5zs//7p4NxfzDTz7QL2PE+a85ZFhJfowJWoSqLYOARcjCM0/s8HHLZxrmi/5BolYIIUSfc+P1j3hL6kGpZRYL7NveNtzd+pNf+TiC9rwZR/l5oBzlr1n2QJpSytp7Nvn8IUN2TVMKlt3Q0tsI3Ll6o7e4GvvsvZuPf/f769KUUtbcu9lbbm9eNiVNyeYNB1/asoIWgfmnbTtKLLMI3Luvvz+NOS9yj542Jo05d/LMo/0yJjLj/CNPObQkP+bWBbd7YRxaXk/98knuZ98tPJzc8YO73WsPHVbMZ0r8gfbC+S36B4naJoPX50uXLk1jBeI0XBlII9hnd3ErMJi3/HjZGPLCstXKCyFEFitXf7yiq0EMbgb1cjXoS25aelpVi2vI1HOv9ZZaxG85fnzDI27oPi/zVt2jjtw3TW0d3jJ2P28NLQdWUxiy3yA/NRCZmx7Z5udZnnq6w7C3dLrKwM57vNT99u7Cm1NE8xnffJ+fFwOHRG2LgaAFc1sAc2cABO3MmTOL+YRJkyaViF5j9erVPo/lrey0adN8mhBChBz/7v29FRU3BMB94Be/WO8OOLC8zyHlDzt8aBqrDlZdrL+NDlbatx/RtZ2I1Y2bnnUfO3NkmpLNe07Y34tlUQARG1pen//jn/30ZXt2WvGB+LNPPpfGSsG/NrS0ZrHjqefTuQLP/6GwniywJiN4sQCL/kOitoXYuHGj72w2f/78NMWVzAOCFl/dkCVLlvjOYTGDBg3yQnbo0M6bzujRo9M5IYToBHcC/GTxo917r88W/WHNHSHm1FOucnvvvZv7yLTarJzfWrDKi+TZF9anw1RfQccwhGtWx67vL32wqpVWlIIf7OwT5vmOXmZ5rSQ2zYpr4FtLZykE6Ds+WP5cO/TdB3jhHC4fdhozyKc+8nBXiIW16FskaluItWsLvmahCIVhw4b5KaIXRo0aVeJOYNbYGKuH5cKyliaEEAYduD7x8euKnboQtKMOv9SL0RgELQL1B4sr+5Ua1HHRhSu8T23oY9to2EgHt910eprSyeObn3W33fGYe++Jb0pTRC0wYgGuBFhpEZOIStwCyhGLTPxyWZ5Ax66ws1kI5fDbRUCzHgKdwWKo3+qDSp3PRP2RqBVdCN0JwhBjYhZRjDWXMkyFECIGV4Kvfu3ENOa8+ESELv5h55BLYIIW4VuLQDVBixW4Oz65/Y0JWkY/OOzgrtbp+x4s+Hpm5YnqYKXFWrvl0e1u590Loja2yhLf7VW7pLGuIFofu+/xNNaVUAATsAizznJY57O4HaLvkKhtMszqaoQW0xEjRvhpbEU1K6xZXs2iW41LLrnEj4+LmJ04caJP27JFw5cIIbJ58skX0rlsRo/8WlHQ1gLWXxO0B5VxY2gEjpt4VVHQlnMtWH33ppbs9NUTsKZmWUARkGDWWARuCC4Gu75y56KLQDzSQTl/W8DnlhCy9paH/FBgcMW5P+ySb769ov+QqG0yGMlg0aLOwZ8RngaiFRHKiAdGOA9z5szxLgSh8KVzGfXGtLW1lXyEgY5j+OQKIUQMw2+FH0pgfFrik993oI9joYVaBS0WWqy/lfxyGwEstLgVMFJBJV/ZxzfvcEeMrL1TXCvzpre/zgvY0FUAQRn61WJ1DX1ew3xEL53ClnzhxjTXeYHLhxjK+dUy8gH5ZnWNO4KNOObNPj8UytQvv9r+RV8UyyEITiyydOgaObJrL1m208AdAJHK1Kyp4dfJGK1gxYoVbvbs2cV8GwHBCL9WRl74RTHEbuhvy7y5I1h9eaJZzhEh6km9vigWfzHs0+eP9R3BELj412ZBZzGG+2LUBDqZIWJxS8Cqu3nzs2mpUqxMb6jXF8X+7RWz0rmuIHQNxp393KeP8iMbxJD3wfcflDk0GFbgffbeteW/KIZgjb8IhpCt9YtiEH4BDMHMuLdhnaTRWcyYdcM5JYIVQRt+UQxBy1BfjQz7sJnueRK1wu+XvIrQeqNzRIiu1EvU5ol6fiY3T+RJ1Ire02yiVu4HLYR17Ao/jmDjz0rQCiGEECLPSNS2EPjUmjsC4pZgH1oQQgghhMgzErUtBhZZRGwYhBBCCCHyjkStEEIIIYTIPeooJkSAzhEhBKxZ39mzvpX44//q96+VOGG/zzfVPU+WWiGEEEIIkXskaoUQQgghRO6RqBVCCCGEELlHPrX9BJ+QHTUq+/N7RrkvhNUb9kNPP7bAGLcMCZZFT/Zt/EUyGMgPQcinVggBfe1TO+Ht33Cz5rzb7X9g4QtWcNstv3Sf+/g1aazAnkN2c8t+/tE05tz0D37f3buq8EXHOK8e5MWn9jcPbHGffv9305hzbzl8uPvcwlPSWCnXf+cu9/T259xHPvuuNKUA/qQxF33/g+51b+08JkZWWeOK9o+6Vwwu/YId62z/8f3uW8vPTVMaE/nUih6BWOXEscDnafn8bJjWH4K2XoTtJsyZM8cLQj7w0B0QtIj5sC59CEII0cwgaJ/Y0vUTv08/+bw7eNRwd9djXyiGULRe8rlb3LbNzxbzBu+9mxe5rYYJWgToDY9+3ocnk/35hak/SEt0gri86tKfpbFOnt62w08RpFYHIUvQQljGApw2/R1dBC3ty1qn6HskakVdmDFjhhfqZ555ZppSHazXkCcxL4QQPeWR+7a4w/f9XKaghfvvftwdcOg+aawr1y9e5627xnlfOMZbbbdvLQi0VuG3D271ltlQgH70whPcg3etL4pV+Mj4b5YVl79/5An3yr126yJIa+VbX/qJb8O7P3R4mtLJN86/wdct+h+J2gaDz9bySj4kTkMMYhW1QD7TkDA/ri+G/O6UL8fkyZNde3t70VpLXbgrhHWHn+jdtm2bGzZsWBoTQojmZtH8X7gvfO1k9+PbP5amlIIV9hWv2jmNiXIgJMu5Ghirbv2Ve9WQ3bxFFfEZ86cnn/f5PQFL7PKl69yEj4xOUzpB7FLvuPcckKaI/kSiNmeYb669suf1PZ+6DUE84g5gr/OnTp3q07IYP368Gzt2bLEs9REQyt3FLK7bt2/3U8D/1uq2T/Sa6F25cqWfhqK3J+sVQog8cOl33++OOuaNaawUrK1YcBd+7XZvzbUQ8u7JB7nZM69PYwV3BPxqB+3VM2tjM/GLn/yyxPI66ug3VBS+j9z7uHdZwKfUAkK4FpZ9a2UXSzGY2K0muEXfIVGbMxYvXuxf85uAHDp0qBewBqIQX13cAQzmsYiGVlJj/vz5PhjUV0/radg285U10WviNhTUCHQJWyFEq/Gnp//sp1M/PqboM4uIDYUt7gYjRg4rEbz17iiWRxCjiElcEGoFQWuWXAL+uV/95DVVhS3uDbg5ZFlp8fP9xFdOTmNiIJCozRkrVqxwbW1taazAkCGdT4ubNm3yLgCh9ZOAYNyyZUtaqhNELOALG5alnnoQts3A7QCWL1/u1q8v9OIFE+gLFy5MU4QQojVgFASEbGjJRcQCoyIAQvZVg3cpil78b0lrNZ/aEEQoYpQOW+U6eWXBqAShRZVlx088yN1+w0NpSjZr2n/tLcLxunA7YHksxGLgkKhtQrDkmvUzDKH11jAxi1i2cj211FrHr0GDBvlpT8gSwUII0argXgAIW+bfd3pnx1rmGS3hjuWPpimthQlarKNZHba6yysG7ZLOlQe3hTcf3vUeiaWYYK4MdFB7amvBvQG3BNE/SNQ2GFmiLrSa4v8aW1FDCyxWXARqLfD6f8GCBd4/N7SYYqntCbhG4Ppg1t9qIKZjlwi2pacd1YQQIq/88MrVfqivGPxs93hlofNYuVETWhGG6jJB213rKC4EWWKTsWxfUcU/GdeD/Q7YO411Ym4MFrAcY9FlvjsWZNE7JGobjMGDB3tRaVZPE54GIwwQD/PDjmJYY1k+HFrLRkuIBaSJT3MHADqO9QT8YGlX6J9bDSzK4YccaCfbMmvWrDRFCCFagyPH7+dFK+LWYAxaLLG4JphbAp3DDMoypBfLthJYaLGE4gfbk9f9dCajoxdDbxnml/vuDxyWpnTFhgt79f57+qloPCRqGww6gOFXyggHCFEss2FnK/KxrFo+rgLkhy4DuBAgMMkn2GgJWR81sBEJrOyYMWO82Kxm7bXyFhCjrLdWKy0ggFmX1WHt1Li1QohWgxEMGOpr/tyflXQEY8QEAz9axqq1fMqyTKuNfmB+r3TMstf9Fmp91Y8/LR3FbDmsvuGXwbAEM85tyLN/KHTm6+nYtqLv0WdymwCspHSuCl0IRM9o1nNECNE9+vozuY1KXj6TK+oDgr6Z7nmy1OYM3Apin1OspIxFK4QQQgjRqkjU5gzzWbVX9gTcD7JGNhBCCCGEaBUkanMIbga8LrAgQSuEEEKIVkeiVgghhBBC5B51FBMiQOeIEAKeemJ2OtdazPr9/6RzohX4zpgvN9U9T5ZaIYQQQgiReyRqhRBCCCFE7pGoFUIIIYQQuUeitp+wT9VWCvbp276GdcWfzK0VlovbbaE3sO3x+LtCCNEsvGrwZ4vhU+ffnKZ2Mul9VxXzDz38a2lqeagjrLOVefqXW71v6J+ffC5NKUBaGMqx8fZfu/aZS9JYeSgT1/nI0rvT3M52WCAu+heJ2n6CT7/ijG2Bz8OOGzeuJC1Pn4cN201grFyE7caNG9MStYOg5RO5QgjRbGzessOLzlmfGeue3PYlH666eq27fMGqtIQriljLb2vbzYvccrAsdVj5004dUZMQblZu+ejV6VwniMqDpo5xH7r9Mz687rgDfFoMgvaO2denscq88MQOd+SsdxfrJOw/8VCfh6CmHcd841SfzpS4hG3/IlEr6gJj5SLU+eJZd6C8BK0QollZ8K3V7oi3DXdnTev8nVtw+QT3gx/c5+dvuPGXbtPmZ92SH57m43DxRce5O3+x3gviLFiWOoyLLzzW17F27eY0pXVY8/Xl7j8G7ZrGCpiQNMEJh31svJ8iYg0sr7UKWkTrC9t3uN1fNyhNKeWXP7rHDT5oqHvFG/fycabEN/z8UR8X/YNEbYMxd+7cLq/h47TYlYH8+PV/mF/ttT753SlfjsmTJ7v29vaitZa6YneF0O2BcgsWLChaeoUQotn4RSJOR44cmsYKnHD8G93dd33cz99z7yYvekP2HrKrt8AyFeVBoP7mpvvd6JnHpikFEJRYSyuB8MXySjmsuNX4rz/92U9f+qpd/DQG0TxuzqQ0JgYKidqcYa/qV61a5cXghg0b3MyZM9PcAohHRKK5BkydOtWnZTF+/Hg3duzYYlnqIyCUu4u5T2zfvt1PYdKkScW6lyxZ4uMmeocOHerThRCiWcGCetABQ7x7gPm/hj61m5N8RG/oI1vNleCUUw50085alsYK/rUwYsTeftoqYGXlNX8tmIXWLK0I3/f8oPY3i08/us1bhEOfWazE5WB929ZtdG987yFpiugPJGpzxuLFi/1rfhOQCMPQyokYxVc3/HQu88OGDcvsHDZ//nwfDOqjbL0I2zZx4kQ/DUWvEEI0O8ef+G23dMmUog8s/rChsJ395RXukIPbivn41FYStrgy4KNrIhhrMMu1ErgOYGG11/2VwHUAAYyPbTlLazX+86nnvfuB+dISsBLHwpZ1IXhZH+3r6fpEz5CozRkrVqxIfvDa0liBIUOGpHPObdq0ybsAhK/8CVhft2zZkpbqBBEL+LaGZamnHoRtM7Zt25bOCSFE84P/a+hKcON1H/bC1nxm6eiFS4KBfy0WXvxts0Dwbt36XFEEz5xxlBe3reJTixUU1wHzk60EInPZ+y73/q2hj213YV2xSwOdxhC2IYhYE73w41M6jUai75GobUKw5Nor/zCE1lvDxCxi2cr11FJrQ5INGpTtSC+EEK3IoFf9RzpX4BWvLI3vtVdXa17b3rulc6UgXBG8dA4zEMQI4+tueCRNaW4ea3/YW03NDcBGP0C8hkNsmaDFYtoX/q4v2f2l6Vw2uB7QTtoh+geJ2gYjy7IZWk3xf42tqKEFFisuArUWrKMW/rnr169PU5231PYEXCNwfTDrrxBCtDp0Alt3f+lbsqefesFPsd7iT7t6ddehEBGusRgOKTcyQiuAQDVrKMH8aif88KyiNZaOYCZoa7HoVgOLayiY4f/98c/FkRdwh4hdEaxzmeg/JGobjMGDB3tRaVZPE54GIwwQD/PDjmJYY1k+HFrLRkuIfWpNfIbuAHQc6wn48tKu0D9XCCFanY9/9AjvMxu6BuBji2UV8I9l+K5w3FrGqMVSm9XxizTyPvXpm9KUwrBguDOceML+aUprg2UU6y0+tPUQtPCaY97i1i28vWh1ZYrf7AEfPMLH9x33Ju+KEI5Lu3LOzfKr7WckahsMOoDRuYoRDhCiWGbDzlbkY1m1fFwFyA9dBnAhQGCST7DREqyjVoiNSGBlx4wZ490Xqll7rbwFhDXrlZVWCCE6QYTes2a6F7LWsQtBG7oP4BfL2LOWv2nTs8UhvwAf2lD0kkcZK89ICPjpttroB+V4/Be/8VNEqLkoWIitreWgXOgPiwUYkYz1l3qY4lM7dMzrfT5T++CCrWvPA4fWTVSL2tgpESItM6YS4qsZNxcr6cKFC0tcCETPaNZzRAjRPZ56YnY611rM+v3/pHOiFUB8N9M9T5banIFbQfxxBKykjEUrhBBCCNGqSNTmDPNZDV/9436QNbKBEEIIIUSrIFGbQ3Az4HWBBQlaIYQQQrQ6ErVCCCGEECL3qKOYEAE6R4QQ8JvNrfXZWePGp/+WzolW4NMHf7Gp7nmy1AohhBBCiNwjUSuEEEIIIXKPRK0QQgghhMg98qnNCXy+tr29PY11Zdy4cW758tLvTvcFzf6hB/nUCiGgr31qx73t6+6iS97t3npQ9lfApp56tTvhPW9xRx/7xjSlAMtt3fysn594ygh3wexj/Hy9yJNPLf6gxjvPPsqNfv9haaxAmA8X3XtBOlfg4Z896pbOujaNOXfGwimu7U1D0lhXNj28xV0xdVEaS/b/7JPcm96xXxpL9t3cW909196XxgocctKB7vgZR6exxkM+tWJAQLBy4hE2bNjg0/j0raX1h6AVQgjRe0JhmgWCdvXKroYD0g8fNdz9etMXfbhr1Xr3xVm3pLmtxSUnzfNCFqFK+Ollt7mV31+T5hbEWpiPuAxFLmURtJ+87hyfj0BFsD77xI60RCmkk085yiOAWR6ha1AmXCehkQVtMyJRK4QQQvQDD6zb7F7fdkFZQbt1yw6fnyVoWZb0D0w9PE1x7qPnvd0t/cHaNNY6YGF9ZtuOEsssYnLtDff7eROaYb6JS5YFRDACdbc9d/VxLK6vPmSY+9Xtv/HxGNLJN8ssFl2E8sPtv/Jx+P09G9yQ/QenMTEQSNQ2Gbw+X7p0aRorEKfhykAawT67i1uBwbzlx8vGkBeWrVZeCCFalW/N+4W7ZN57XPvKj6cppXxv4V3uE58a662wMbgpkL7XkIIIa2UQllhBy4HgrJRvonev1w/yU+MD33hfFxeGWjEL73+8/KV+KgYGidoWA0EL5rYA5s4ACNqZM2cW8wmTJk0qEb3G6tWrfR7LW9lp06b5NCGEEKUsvPrULj6yIfjHfugjI9NYdc4758deBLc6CEosr+/4yJg0pStmoUXIPv+HP7uXDd7VvfCnP3uXBAuhK0HMG8a8zltirR7K4j/b9paCT/TWX2/3dX7lxHnF+r730R/6PNF/SNS2EBs3bvSdzebPn5+muJJ5QNDiqxuyZMkS3zksZtCgQV7IDh06NE1xbvTo0emcEEKIvgDfWtwUoDsiuBnBtxYhiaAMO22FIHrxf8VFwdwNcF+447urir6v5lNbTtiyHP631INgpSx+tbbO55963tdpPrqEZ7Y9K2Hbz0jUthBr1xZ8r0IRCsOGDfNTRC+MGjWqxJ3ArLExVg/LhWUtTQghRP3B4osrwg9+/CEvbvG3bVXOu7YgIrHSIjbNDcAgjujFHzZ2LcDdwECc4iOL0M2CjmXUY4KVgLBlxAOgbtJMNMPJFxzvrbtxm0TfIVEruhC6E4QhxsQsohhrLmWYCiGE6Hvwsx05erh78P6taUrrgijFWosbgGGCFrEaCligbMyur9olnesKndCw5oZgqY2H8AqRf23/I1HbZJjV1QgtpiNGjPDT2IpqVlizvJpFtxqXXHKJHx8XMTtx4kSftmVLeZ8kIYQQPePWm39ZdDkI2VxhaLBmBaspbgcxvP43cCMwQRsPq4VfLWVjC+qOJ58rsbTG4GJQDtwMzGpr4LMLleoU9UWitslgJINFizoHh0Z4GohWRCgjHhjhPMyZM8e7EITCl85l1BvT1tZW8hEGOo7hkyuEEKK+WAezcFza73xrtR8erNX8aum0hSgNx6VFUJpfLWIV1wB8aLPGiUVkIna/e+4P0pRCRzKsrm8a94Y0pZQRJxzgO6OFQph1UA+89Zg3++VDn1xrg+g/JGqbDD7CQGcw83GNO27ZRxosH7DuDhlS+IrKjBkzvLAlLSyT9QUxyoKVwxfXrL61WnuFEELUhn1wAYst4asXr8gc/qvZQZTiv4rItJEGHrt3g/evBRtrNsy3YEIYsYtQtXQ6gIVfFIutwfjMIlCx/toyoRUYMW2dzSyf8j0dIkz0DH0mV/j9gi+suRC0MjpHhBDQ15/JbVTy9Jlc0XsQ3810z5OltoWwjl3hxxFs/FkJWiGEEELkGYnaFgKfWiyy+Mwibgn2oQUhhBBCiDwjUdtiYJFFxIZBCCGEECLvSNQKIYQQQojc03IdxYQQQgghRIFmkoEtJWqFEEIIIURzIvcDIYQQQgiReyRqhRBCCCFE7pGoFUIIIYQQuUeiVgghhBBC5B6JWiGEEEIIkXskaoUQQgghRO6RqBVCCCGEELlHolYIIYQQQuQeiVohhBBCCJF7JGqFEEIIIUTukagVQgghhBC5R6JWCCGEEELkHolaIYQQQgiReyRqhRBCCCFE7pGoFUIIIYQQuUeiVgghhBBC5B6JWiGEEEIIkXskaoUQQgghRO6RqBVCCCGEELlHolYIIYQQQuQeiVohhBBCCJF7JGqFEEIIIUTukagVQgghhBC5R6JWCCGEEELkHolaIYQQQgiReyRqhRBCCCFE7pGoFUIIIYQQuUeiVgghhBBC5B6JWiGEEEIIkXskaoUQQgghRO6RqBVCCCGEELlHolYIIYQQQuQeiVohhBBCCJF7dupISOdzxU9+8hP36KOP+vmddtrJT4UQQgghBgok1f/93/+5V7/61W78+PHuJS95SZoj+oPcitqLLrrInyxveMMb3F//+tc0VQghhBBiYPiXf/kX99BDD7kXXnjBfeQjH3G77757miP6g1yL2iOPPNIdfPDB/snIyVor6kUurwghRH+ju46I2elFO7k77rjD3Xfffe7UU091r3jFK9Ic0R/kVtRefPHFbtSoUe7www93Hc8+6zo2bXbuH/4hzU2o5dcmq0y1tGr11pyfUbDSsuXyMtPTxG7U16VoN5YtwedVKJCZlSZm5fUmLaQ7y1SsK8jsTp0hlcqUyytJzygUJHXJranOgDg9q1y5ZY1iflSw2nJQqUyP8pKMHtYZ/zh2+bEMEsr9kGald62nkFKuDqi1/sxfdLYxSa+1DiMzvUw9pKWr8cRlMpfJSMwqB+XSjS75SULWMh0VaiqX49MzMiuWz6BYT3TO2X7IWi4zLSsxpSSvB8e9JC0q0J16oGz9ZRaIk8vVC9XrCf+WEqaVqaIzPShQrqzxfx1/d227Dna/uesRd8+6e72o3WOPPdJc0R/kWtSOHDnSh/9btdr99fL5bqfQdyXsAmc/IF2m6YzFIS4DyZOXp1I5P03+xOmQzvvVFdOTGYsX0xJsvkt6GonLxnE/TWbCvHgKfj75k6YVDd3hlMDZYfP88dPCbMm84dPThDg9nEJYFny8MGvTznZF5cpNi/M2k2DpYT7YfLk2QLGMTTsz/awFIy5XjKfB9idYGmSVN6ycT0szLN+mYFlM/bwlFCYlZbk+ivFkxs+nU0tnSnvtWvLp6QaUa2/JNClrjUkn4CfpfHEKlhHnZU6TP2G7Ss7TeJr8sThklkkmyXXOzyFVGcz7ePLH0otpTG0mgdm/F2YL+Wkek2II0mzq59OMLulM05kwDSw9XKdPS0JHsk3MsmlMfZnkD/OGL+PLl25zvA2+CPPpNExPZ4tpwaRYDwnFMuE0WW/JulJ82TQhTP97Rpovm85YuqX5YIkJNvv3aHttPqu8BSuUlZa5DUnE4uG0cLtl/XbyWXqhHisL1pbCEp34ePLH56fVWH4xnXn++GOcrC9N5G+aXZgmfyytJD3F18e0EPVYmmH5mWULDUimnfu8OE3LG2F+WFcx3WYSinkZaVBMTgoUz8MESw/PpeIyGWlgbfHzaQA/DfL+nsz999/+4k5+/dFun627urvvXuMmn3qKe4VEbb/SFKL273ff7f56xZXOveTf0twE+83o/O3omhbfmCEuA7WIWqC+MmWKycz4SJpSjAdTCNOhXFvjuJ8mM8X5NBhhukWSiVVfvixQsDDxdMlP8PlBQnfLRvlhdjFiaVHZ0nhG2S5lbBokli2TTiEtX1yMablyJfFoCsW0dKZcGQueZMbmi2kJ6XxJu8KyYPMlQpVpUKiYlgYjTLdIpTLF9GQmiPuJ5dkULCPOi8v4ePInzrcplKQlf6qWSSY9ELWQdTP00+RPnBaWtamfTzO6pDNNZ8I0sPRQVFka2KxPT2e6pEGyUHE+oVhfVN6mYXtKplE6+LqShDCNeR+C9XaZpjMWh26L2jQhTPckGeE+M8ot4+eD9Dgts64kUhK3YCtIsLlCemG+S13JjM8vxDw+nqYbNh+md6aV7mebBytfkmbTNM/Ph9MovTifplsc/Hy6fksvpEXxYAoca+JhGvX7aRogTOs6Tf4m/7OOTyVRC+XSKx0ftvK///Y/7t2vG+8Gb36pW5OI2vdJ1PY7oT0zx4R3qlrJWCarmp5UXY64rr6sG2pZX81tiAr2dH210qWu3lTWTNRxP2RVVVP1DXAs6t2EPtykelbd33X14W7JpDfri5ftzU9Gl0Wz6upNYyNoa6Cd6roferMj4iWzaqqldsrUUldMufXVVldpaq111UL5ZXpSm6gnTSJqhRBCCCFEKyNRK4QQQgghco9ErRBCCCGEyD0StUIIIYQQIvdI1AohhBBCiNwjUSuEEEIIIXKPRK0QQgghhMg9ErVCCCGEECL3SNQKIYQQQojcI1ErhBBCCCFyj0StEEIIIYTIPRK1QgghhBAi90jUCiGEEEKI3CNRK4QQQgghco9ErRBCCCGEyD0StUIIIYQQIvdI1AohhBBCiNwjUSuEEEIIIXKPRK0QQgghhMg9ErVCCCGEECL3SNQKIYQQQojcI1ErhBBCCCFyj0StEEIIIYTIPTt1JKTzueLiiy92I0eO9OH/Vq12f73scrfTv78kzU3YKZpCSVryJy5jU7B5L/vTSK3lgNmovJ8U04PEdLY4hTAddkojcdl4CpQlHgaOss2Dn6aRZGLVF/OhJC2NMLFg2Hxxms7E6WDz7C/fpiQhLBeVLbbLRzpnS6bAfDGezMRlLD9OB7+SJOyUNIhZ2mWPe13KU64QKbaNaThvOzvcNigpE0yhUlkLxXYlEcsHm0+nJe0qV5Z6StLTSFw2jvtpUJaQtb/A8vlj6TYbxItYRpwXl/EhSIzLBVm17C+b7vSinRw/h2yOwbyPJ38svZjG1GYSmP17YbaQn+YxKYYgzaZ+ngz+p23pTE/++5kgjTKkM5/8YZ1+Pp3G6wDfriQhTPNl/UzpNofbkDariE8jBInl0sDWG0yC8p3rjfMsoSQ9I604Tf6UtDudKc4HUyoKyxq+bJoQLuPng/RiWlIJ9dgxAyvry/hIAR9PyvnbbZpu2X75JPL3zqzilBnm43SrJk6HrnnJvzQSpkNcFmzep6eRrDTwcQtpusXBT9MMSy+k8SeIB1OwfRGmxfUT+MnuctxTTNpYPlg+9Ru2XEdQ0NK41nw1PpKxrjSdyd+Tv//9t/9xJ7/+aDd0227u7rvXuMmnnuJesccehUKiX2gKUfv3R37p/nbd9c7967+kuQnBD40njNt8PA0J87JuoEYYp1xJPJ0m+FmLhxEmBI5CSX4whbANkFmmyhRK0gqRYLZzCiXzQYTZsuWKfyqXIRS3Oc20dCOZL0STgpVEDJTMlynb3fIhJelpJJmEUU/JvBVICefBx9OdwHxW2TCdoonw8oRlIYzbvJ8mf+Ky0EW0ZxQiKUwumSdCg5gvTDzxfLRvi7G4XJGgUDyFYlqQmJmfTj1JJCs9SqPK8MZp2HzmNCjIbFZeSXqQZhTmO4UHMBuWgTjNynOjZRN8flCgpGz6x9JKpulC/LV0CMsQ4nVYPli6lQGfZjOdk2CazBX+F9Mga96XCTMSwjz+ZIkXlikpB0lil7QE5sM4FNPSjLhMWD/YfNjWYlqyd2yb2VFBkWL5LtuQppeUJWSkg48nf3wZ5j3puZWuszM9mU8jJWnplBnmu+Sl6UaxTJoe55X+TadphImlQ3E+mQkFpCedYWJpfhqkG53znQ8wUFwuWsZPk4Kdy6XzdpzSSpgPy4ClcWz/8re/uqOGHu5e/Pu/uTX3rHHvP/X9bg+J2n4l16L2bW97mzv00EPTFCGEEEKIgeXOO+909913nzv11FMlavuZ3IraCy+80B1wwAE+CCGEEEIMNC9+8YvdHXfc4X7961+7D37wgxK1/UxuRe1ll13mNm/e7HbZZZc0RQghhBBi4HjRi17k/vCHP7g999zTfehDH3K77rprmiP6g9yK2scff9w99dRT7u9//7vbKcsfUAghhBCiHzFJtfvuu7t99tnH/dM//ZOPi/4ht6JWCCGEEKJRQV7J6Na/WB9oIYQQQghRJyRo+x+JWiGEEEIIkXskaoUQQgghRO6RqBVCCCGEELlHolYIIYQQQuQeiVohhBBCFGGozD/96U/u6aef9vMx//M//+O2bt3q/uu//svH//rXv7onn3zS/ed//qeP1wLLbNu2zdclCvv8ueeeS2O9g1EXODb/+7//W4w/88wz/pg2+4BXErVCCCGEKPL888+7M844w73nPe/JFFp8BvZ1r3udu+GGG3z8N7/5jRs5cqT78Y9/7OO18Pvf/9695S1vcStXrkxTWpuHH37YnXbaaWmsd/CAMWvWLPfQQw/5OAL34x//uDvrrLOKDyLNSkOIWp4gfvKTn7iNGzemKaLe/OUvf3EbNmzwT8e9hSc9PnzBUx/83//9n3vwwQfd3Xff7eNCCCHyC0NR/cM//ENxPoavZoVldtttN3fSSSe5oUOH+ngtWL3/+I//6KetDvfPu+66K431Dr5otmjRIvfv//7vPv7P//zPbtSoUW706NFN/zGIf/h8Qjo/YHAw3/nOd/oL4uCDD05TRT3hoeHyyy93Rx55pPu3f/u3NLVn8OQ+c+ZMf6G8/vWvd//93//tpk+f7trb29373//+tJQQQog8gkvAzTff7N0JJk2a5P71X/81zSmAgWTZsmXuuOOOc294wxt8GiJ12LBhxc/CYki59dZb3Y033ugee+wxn7ZmzRqf/spXvtL98Y9/9MLr+OOPd7/97W/dNddc4x599FH30pe+1L3sZS/LFNMGxpl77rnHXXvtte6OO+5w/+///T+31157lQhkjGQ/+tGP3IoVK9yWLVvcy1/+cl83YIhZt26dd6HgFf3ixYvdL37xC39vGzx4cInw4/7205/+1LcP4w1C/hWveIUX9oDbABbR66+/3t1+++3e4MP2hfvse9/7nk974IEHfD3333+/b6vV8/Of/9wtWbLE7weWY/uYrl271j377LPupptu8vuOT+/+x3/8h99P7Nvly5e7++67z73wwgtujz32cC9+8Yu91fy73/2u3z9/+9vfvNX9zW9+s5+nzJAhQ4ptf+SRR7x1nW3Hust+NyEM7D/0Asebcmg1jt+rXvWqhn0YaQhRi1/N0qVL3dvf/nZ3yCGHpKminvzwhz/0F9TEiRPdS17ykjS1Z3ABffazn3Xvete73Gte8xp/gXBxHnDAAe7Vr351WkoIIUQeMVGL4Dv11FO7WPfwtf3BD35QFLW/+93v3Ac+8AEvmHAp+POf/+zOP/98961vfcsLToQW9/gFCxa41772tW7EiBFe1F511VXeDeFXv/qVF1UIVAQcxhLqygKRecUVV7jZs2e7J554wi+HIOTT+bhAYGzBPYLX7Qi+HTt2uNWrV3thyroHDRrkt+sb3/iGmz9/vhek69ev99vANvEm8sADD/Sije386Ec/6tvONlEf4g7xR13c+6jjwgsv9GKWtiCiEZTcD3fZZRdf3zve8Q4vrG+77TZfbtWqVV4wDh8+3O27775e1FoeohXxCqybtm/atMmHt73tbV7Q4kaAbqI84pz2IWgPOuggv695kGCb/uVf/sWL4zFjxvjjgTA+5phj/PHkgeIzn/mMrxfLLoKc/Y9xkfXTboxU1E86+xpRf/XVV3sNwT5qSJKGDzjJU0JHsuM7vv71r6cp5UlOlo7TTz+94+ijj+5ITtqOZIf79OTJpiM5sB2zZs3qSE5inwZz5szpSIRcx3/913/5OOWSC6Djy1/+ckdy4fq0mOSJpqO9vb0jOaAd48eP70hOho7kqS/NLZCcMB0f+9jHfDtoT3LQO5ILJc3t6Lj88st9HYTTTjut4+STT+742te+1rF9+/a0RIHkgu/49re/3XHsscd2nHTSSR3z5s3rSJ7M0twCK1eu7Dj77LM7kh8QP03EaZpT4IILLvBlkhO5433ve59fV3KhFeu58sorO5IfiI7ddtvNb88NN9zQkTyRdiQXdcdPfvKTjunTp3eccMIJHcnF6Mvfe++9HcnJ3pE8QXeceOKJHV/4whc6kgve5yUXjN+e5IejI7loO9773vf67U5OdN/2EOqZNm2abzf7Krmg0pwOfzw4BsnF7dv9nve8p+Pd7353R3KhdSSiOS0lhBCiv+Eeyr0kEVj+3sQ9hfuThUQ8drz0pS/191xIhFbHa17zmo7vfve7Pp6Izo5/+7d/60gEYEciBjsSQdfx4Q9/mB5K/t4ELPPyl7+84+CDD+54+OGH/X2E+8GwYcM6EtHW8fe//92Xi+E+xXLcl2kn95KvfvWrXkNwj/nTn/7U8aY3vcnfmzdv3uzvsYlw9mmjRo3y7WFd6AfqQXdwP/zP//xPf8/feeedfXvhS1/6ki9zyy23+PUk4s/Xm4jyjkTAdjzyyCMdiUjuuOSSS3xb0A7omUTwdnzxi1/0cbaD/ch2cU+kHrY3EaAdEyZMKOoVtoF7tOmSu+++u2PXXXf1x4H1sl3Uddhhh3Uk4rYjEbUdicD3ebQ7EaMdv/71r/2y6IFEUHfcddddvg2sc8qUKV4L/eUvf/H7fq+99vL35a1bt/p9hIZ585vf3HHKKaf4NrAu7vH//u//3nHrrbd2PP/88x2JAPaagO1nvhHJVUcxnhCSA+N9cA899FD/5MCrEV4d8FTF6wrM/Dz5AU905PEUY/66PG0RxymbJ5sYTPTJSe4+8pGP+PqSE8g/wSQH0i8DPDklItC/KuBphWWmTp3qvvnNbxZ9VnnqwpqZXBRun3328a8ekpPWJSe/f3IFXgtgOb300kv9Ux9Pponw9a/2eYoFniaTHwP/uoSnW16PsAyvMAye8nii44mR1z+8YkguKB/Anrx4jcBriN13392/QuApmSc12sH68YviCZJ9nPwY+G3jVQxW3k9+8pP+KTS54L01lidA1sXTKNuM6wFP2AavP5KLw786oR62hyd59gGwfvYj+42nYzod8EqDbf/c5z7nywghhBhYsEbGoZJrQKIr3MKFC711kPsmr7NxDeC3njd6WElDzjzzTLf//vt76yFWXqytWA7tPhnCPZ1X7lhbqQ9LKJZILJdYE3lzyKt9rKKJYPP3VO57WEPPO+88b5XF2gq0g/si6+e+hvWR+zr3Wu5P3Ne+853vuKOOOsq/lWQ93DuxNuPKhzvD97//fe+igcWa+yjuDNxL2R7cEbZv3+7XhctC8nDg7+HU88Y3vtH7t1LethM9wn41XYJbA+4SWGdZr7lkoBdYL23nPkzeEUcc4TUQ+gZIB9Zlfs8GlmyzwnO/5R7PPqIO9in38kR8+7LsC6zM7AMsyHvvvbc/pliwEzHuyzQcBW07sNRiqU0EZMcb3vAGbxXlSYunCJ6ueIrBCol1j6cf5pOD7pfBqpuIJf90iDUUeJLhKcqeKGPI5+koOdhFy+vatWv9U+hll13m429961s73v72t/snLCy/PAlRnuV4EgOeiJKLxD8pUQZ4skxOzGIZtpen2TvvvLO4ruSC6UguQL8c1uG2tja/PWwzJIK+44Mf/GBHcgL6JzTg6ZOnqV/+8pe+LeyLT3ziEx2J+PRPkpAI7I7kQcA/XUIiMv2+Ofzww33d1kae+Kibp8DkhPb7mafx5ILxFm6gXcnJ3ZGIUV/GngKTE9/nY8VOLmpvVWY9lOEp85xzzukYPnx4x+OPP+6fDHniZb9ibWc9PCmzbclDQBeLthBCiP6Bextvz7hnmLUTSx2B+8t1113n7zmxpZY3bVgPsQLy1pTfdYP7ApZAuz+bpfZnP/uZjwPLfuhDH/IWTNYZwz2fPN4sZsG95sILL/Q6gPtMCNZarKq0nfst1mCsxCGJYPYWzscee8xbK7mHJ8I2zS2FbeNNZSLyfb2vfOUrfUjEZkciTP19jDeblOO+H77JJA3NwP33iSee8GnkU87Aas199JprrklTOuHe//Of/9xbvXnT+vrXv95rhUSw+3y0D9th8dBSC7wVRcfEb0XZfvRRInp9G7FuoyVMH8CyZcv8utBbjUhuLLWJ2PSWWYYY4cmPJxaervCbwRrIk0eyo11yEPyTGvC0wRMavrpYIAF/E572eGLKIrnA/JMMQ2uYHxGWRurCYoq1FqfpRFz7p0SejngSmjx5sn+SwmoKtA+rKJZeykByEvknVdYPWEBpB70SbV2JqPPO4SyH5ZNt5kkqEazesonfEE+9+LYQAEsxT5L459AWnu7oEMYTpK3LnqztqS059v7pDEsr+9PamAh3d8stt/gnYXsixzLLE7A9UVodTCkTgz8RT3IcK55mKcO+mTZtmvcB4ljxNMqTIk/z++23n18PT8pjx471T8j4dAkhhBg4uC9hoeNewZTA/cU6XMXwO87bRCx8dp/oDtyXCJUgP+u+w30wEWL+/kE74jLWnqxlDZYP1089ZvU0yGddNk8/ICzAdk/GEoy/K31Y0B9Gte3KgvXH0DmO+zGag3m2hzZw/6x1Hdx7WS6uHx2A/skzuRC1HChcCng9zqv1EEQsBwYRxAXH6wscpLmwEII4nNO7EjGaPK34Xn6IX4RUFrg2cHLwOtygfgQ0QgxxzYlPHSG85sDFgNcWwDK8FgjhouJHwk4k1oXJP7zIOKEQy8A2UJ7XI8mTlTv55JO9eE2e6Px+CC8sRGNYj52wYVoIy7Au9mkI+xDXAxzv6UOYPDV7NwLEtQlvo9wFRE9X1mvbYVC3jZZA21ieH0cT1GDttn0khBBiYOA3Out3nntgFohZDDfcL7kPh8vyu8/r8d78tnOv4F7H/chc9IC6cYfjvkXHNe71lAnBoEW63cfK3b8M7kWI+XgcXTphYbBi+8h/9NFH/b2U+78FXvvbvaye0JkN49p73/teP48R7itf+Yo3aLE/4vWVu/+jkzA8IW5DcJfAdSPPwjYXopYDxYnIhRRfTCbsLB3LJ2IRn1ZOOsQpVkuenBi+AnGLNbAcrCvrZMf3hXq5qCgTt4OLmbRKF0pWnllSDU4yTirqI2AxpWcoFk4LtAORf/TRR6dL1Y/rrrvOW08/9alP+fViFcYqjYiOt7kcHCu2NS5P3ES/EEKI5oP7I31dGHKKey73Me5zDCvJW87eCCYMTtyfEHTUZ+C/imDFMspbTtZBHxyMXbZ+3kIiQumPUwvcx3jbiA8vWgK4r7Eu2sEbTt7ookHo+2L3NoxCGJ/oQ4KfbXeodo+0N5gYwzAIUR4tYMI7FLG0lbe1MbQX7cA+YQSGF154wZdlG+mDxJtpfILzSi5ELWBuxzEZwRfCK3kwqyBPa8wz7AYHCssgwpZXCKRxAowfP96XzQKLK0+T5igNnDhYK+fOnestvFhcEXwhXGQMjWHtYN3VwNrLkxYXncErC9wluHBoC3XyhBlChy0GaeZkrBXaQ7vjJ7kQnPMvuOAC7xjOfqIzGtZaTnDaYReMbVu510vsc8pQXwhPhtTLOIa17B8hhBD9D7/PvMZnGhsngHuWlbF4eB/ja2S8EmdIKO4hM2bM8J2bQqzecDnqtJC1XuD+iGhGSH7605/29yyG1GLoMQQZBpiLLrrIi94JEyb4ob+4fyOoGQqM+w91m5tCCIIvXC8dsOnAjGHny1/+sncP5N7LekmnExdWUzpGf+ITn/BleLvJupiyLsP2lcG6w/XRoZw01olI5X6LUDfDHWBkwh2QTuB0qGbbmCKkMbjhiglYi1k3+579zltm1sN+xXCGsOcYsd/oZPeFL3zBd6zDUnvuuef65YH2xNZciI93I9EwopYDyBeqEEKMVGABH1KeKHAzoMcgvQ4ReTyxMPoAowsgZLHGAkKQsohCXoHwKgTxxUFkWU5EfFvLQS8/nuZ4xc/rfw4oTy+IOvxPEWyHH364v6DwncHPFOsp5TnI1g5OnmpwoSCG8a2lHsQ0QpL2IsTpZcgJzEVLOdtmeijiS2xf9KoF27+c9OV+LDiBaTcXA094XFBsI6M60CYTsdRF4AKILc3Avqbd7G98jagHtw2OFa9JGCfPnjiFEEI0FoggxOO4ceO6+JQC/TwQlvyeA25suMjZF8UQlhiR8PvEDZA6EGKITrNEcp/BEkovfgPrKJZWRgYoZ9FlXRdffLF3y0M8cm/h4z/cXxB23JtYL6P7IM7oo0I78T+1t5vcyxhlge0LoXc/24UVFrD8oiUoi5jl/sjIDox2ANwzEcroAe6FjOmK/qDfSPgmle20j1QAy+ECgEUXqy9gTGI0It7EYrxjv7AcYtdg29EL5lLJ9iPm2TbcE+0ezXYwihPWXNwj2CdsA/13zN0PQcxICuRhUUffLFu2zI/SYLANPJxQxkBjsS7cQBqS5CANOLfddpsfGy45ATuSHet7SBKSk8OPycYYcUAvSXpjJieD7/1Ir/8xY8b4XoIhjPfGpiUnfbF3Hz0ukwPuezxWIhGmHckJ4seQY0xXxmRLLsSOT37yk8Xx5BKB6fMSgevH8EuErB+3j16KiWD0ZUi30QCMH/3oR75HJL0WIbm4fQ9ItpXyyQXmeyQy5quRCGrfQ5N1MD4g27zffvv5NhrJSed7jCZCMU3p8OPKsT9tbNif/vSnvldm8mPhe38+/fTTfjQJ1h/C+LT0bBw7dqxfX3LB+H3NaAfkAe2m1yc9XOlNSS/V5IegI7kofT5wTDl2dqwYUYFtsJ6cjKFL/LzzzvP1GYywkPxg+p6yQggh8gdjjzPOqt0Pgfs094xyowkIUQ924k9B3g4c9hUOrIhhc5jnaY0nGMZ+A55MGDcWqyP+plhGeXIIwTeE+vjkLlZZnopsOZ4AcWWoBBZXnlxwbcDKaH65YY9P2ozfLr66PLHwGoKnOnuiMbeC0NUBqzNPcjwx0XbAakpZek3ydMxTEe0On4zYHl5HsM083WIpxmJs8JRGpzTqteV40mPcPj4/bJ3K2Cc81dJWnhr5wgqvM/gKiYHFmM50lAN8lLGQs36eXnl6B/Yn49nx1H3OOef4fBuvz8CqzD7kuGIx5ynRnuSxgOOrRLvD7aVenoh56i/Xw1YIIURjwn2bcc0ZvQc/Vu7BvIHFUsu9jHHizcIrRL1pCFErhBBCiOYAo4997ADDDUYLDCm4zplhRIi+QKJWCCGEEHWFN3R0YOINHm8h6RjNW0Mh+hKJWiGEEEIIkXs6HTeFEEIIIYTIKRK1QgghhBAi90jUCiGEEEKI3CNRK4QQQgghco9ErRBCCCGEyD0StUIIIYQQIvdI1AohhBBCiNwjUSuEEEIIIXKPRK0QQgghhMg9ErVCCCGEECL3SNQKIYQQQojcI1ErhBBCCCFyj0StEEIIIYTIPRK1QgghhBAi90jUCiGEEEKI3CNRK4QQQgghco9ErRBCCCGEyD0StUIIIYQQIvdI1AohhBBCiNwjUSuEEEIIIXKPRK0QQgghhMg9ErVCCCGEECL3DJioHT9+vNtpp50yw5lnnunLML906VI/L3oO+9r2aSNh5wDTWhg+fLibO3duGutbWA/ri+fzwurVq/2+3bhxY5rSc6iDuqgzC86tWo9hd+DaZ731ot7nT73q68129tW+bwX68/ekVvqqTfU6T6ij3L2Ec7g79+taf6PYJ5SzUGv5rN8rqyOE/W3phJ7sp0r7JYS2ZdXPsqw769hTnrxweyytN21uVgZM1C5fvtx1dHT4MG7cODdt2rRifP78+Wmp1qXWiySLegqavoK2tbe3uw0bNvhzIQsucH4EhOgJOn96T73EUJ7Jw+/pQNAf5wb7fezYsUVtsGrVKjds2LCqx4Iya9asSWMFsgQ3aTNnzizWT4C+2C7OI36PuO9ltZ8233777WmsgN0nY9avX++WLFlSbHO5e2grIveDFoATvtEeFLZv3+6nQ4cO9dNGZsaMGf5HpFXhGPHDOXLkyDSlf5g4cWLxJiNEK8DvDL83jUp/3ksQzRi8wvXxG4QB7JJLLklTskEIL1y4MI0VWLlypV82JCuN9ZUTnr1h8eLFbsyYMX5911xzTZraCW2O17t27dou7QOMQYMHD05jIqThRS0nXTkTe/jaoFaLDHXYMqEllBPJ0gnhiUXdlA3zQ+xJ3kK4bLhc2H6eEKk3bI+9emAZTu4FCxb4eXvCs7LEIVyWAKx71KhRft6eaCkXbmu438I22XaEbQ6Xs9ekFsLtLAfttvL2pMx6rI2k2/aE2BM0F294bDdt2lSsL0yHuH21UG5bQ2KLX7j/bD4+XyzfthkoY+lxHoR54XGBStsWngfxNvDjb3l2fhlZ5w+wLvJsO37xi1/4aXicspYzwv0TH6Nwf8d5MbbNUO3cjGHZ/jh/ytVHPNxftDVsb7jv+Y0z4n1v51VYPj6OYOdWeB6yjJWNty0sZ/vWgi3DlN8gfouoywjLUm854jbZeizOsuE+C49tuL5a9kmlc8EItzOs38iqj3XFv6fQnbYyH14T5Jdri5UF0sP1EGz9EB+3MA/CvEqwXLh8peNkbSp3blS6X3eXFStWuClTpqSxThCd1YT16NGj/TTcJ7TX0o22tjafHpazh/h6G1xYz8knn+wmT57cRXAbCFiErLFo0aIubba29reRITckB2/ASZ7GOpKDmcY6oXnkQXJz8vElS5b4OFPipAPLW9lyxGWSH6lifWHdc+bM8XGDcgSDecqAtWvVqlU+Th1WNq4n3E5rv63T4kZYlrrDskDd8bZYm6y87Zus9RosF68nqx7bTqszXn8WWdvANIxbfVmwjni/h/Gstlr98bJZxGXC+sK8cD5uN9sYx8ttM3XYMbQ8g+Vs3bavbbl4nZSzfc+6bB5sHbaM1WnHPasOYLm4rK0/bk+4jZZndcXrCdtHnu1HIN3WmYXVBfH2xPskC8qG62M+jGfVZ9sYL5tFpfrYbttHQLlydTPPshDvewj3oe1vykGYF+9PylHelrF9RRlbBsL1hfscwvqBttp2Wb1hW2NY1tpq2x223drLNFwvy9l6yu2TeB+G254Fddi6rU5bplJ9rJey4f7rTlupy+q2fRbHrXy4Xuottx5bLt4eI6ssaeUgz+pi/bTD4tQTtsnqZRrWGa4jbl8W8X6NIS/cj7VibQ/bTT20zdYZQjppBDsu3SXcL1nQnnBfsZ5w21iWEJeztob7gjIsb20mlNuHrUjp0R0gyp0QHKzwouBAZl1cYCdrpYMb12eQRl5IWDZcL4Trth+ALOLlwvXE64zbH66j1m0rVz6sK95vYTvi5ZgSJx3CdVQjXha6u03xvq10HJgSN7LWHxPXF66v3Hy8nnA7qm0z81ZPJaxOO//idYaE5ULi/Vttf5Bn7YzPzXDZrHrC9oXbC2E7bD6rvVl059zMIjxuwHx4vMO2xvu4lvor1cdytm6ry4iXC7cz3vdAPNxnYVvDecrYfLh+oA5rWyVoF2VtP4f1x8cAwm3Ogvosn7K00eK0z/Zvb/cJy4bbG5NVR7jOSvXF293dtsbl43WF8bBsvG/DYxG2z7B6ss7dcNksqK+W4xS2Ka4z3q54u2OyzqeQeBtqhfXSDoLtI9pKW2ydWdh+I1TaV1mE+yUL26cGbQnXwbIEawNTyluZcF+wbLgNLBfGW51c+9RizrdXHfaKqBpZfihbtmzxr5ZC4ngl7NVMFrwCtTZOmjQpTe098aun5ORPcyqDzxavXIzu+OUkF1rJPrfXZFmYz+ygQYP8FPraf5ZXYda2Wo4f2zNkyJA05krmKxFuR7h91bbZnPmtjeHrufD15OzZs9PUTirtu574VvX0/Mnaxphy1yWvy5Ifan8dWD7taBS6e/5Uwl4Nsn34zyU3Hh+H+LyrhL1qDI9xeP2G4IPMNrAM/ntTp05Ncypfu5yHlh53VMmCfWPlWV8lDjvsMP8aGfjtoY3Es16hdve3MjyPWLYSWb/xMd2pr69+17sDx9TaQDBquUZjunOc+guOV9zZC6xNYG4ehNjdgW1gH1Gec59trAS/sYkm8stwXtfrt4n1U198ftm1GkIb+C3GBQHXA3xwY/C5pp3Geeed56eN9Fs6kORa1HKj4OCGodLNH7Zt25bOdcINhhM5JI5XolInouSpqksb6wHCJ9z+WuFHAD9AI2t/lMMuekLy1OgvzHIXkv2g2g8sxBdwveHHwNpnodKPMT+a3OyMcL4S4XaE21fLNnOuWNv4UTNxwb5E8JGe1ZO10r7rzjE0enr+ZG1jTKXr0jp/Ebg2an0Y7Q+6e/5Ug44fiEuEYugXF593lbD9Fh7j8PqNYRsQ0QiS8CZe7tolcB7ye0ferFmz0iXKY2UtVPJvZP9RHr9M9odBG8M4dPe30q4XC5V+h7N+42O6U19f/a53B86juA1cX7VcozHdOU79BevNesiiTWZICn9Ps343uR7wAWZfZV3LCMz4HpZ1zfUGe6gNjxOBNpEXg5BF0HJd4oMrukduRS0O5Dx92c3eLF2V4MTiZDF4suOE54cAuKDDqaVXgpOOHwO7MJjSDtqFpYSbh8G6Kll1Q+zCqgW7MRmVntBtvxnsD/ZLNcLtAltHuXXRfn5QuKkDy9FGnORrpVZrFlAv9dtx4BiG7c2C4xM67DMfWreysPVYvaFVtdo20x47t4AftaxtjC3g8Tpt26DcOd0d4vOnEraNtt20KTyfKl2XzIfnP9vOPugr+vr8qQZ1si+oN/wtiX8XqgnJ+BhTZ1YHGiDd6rabeHeu3fgtQWgVpj6OV9jznHrj8zWG9rONJuwRK7TRrgvo7m+l1Wlw3seWuhDb/9ZWjm8ocivVF++n3vyu1wu779jviR1jptWu0XLUcpxCyr0xqBc2CkH4e8b20abweqgEApE6yglztjl+sLZ9Wsv9vxa4r8SdvSC+/xg8jNJmrrUsHcD+CM91rkfKZon2liR5Yhhwkgsw0x+F5uFXYiQHrsRHh3nKWEgu8jSnPKzLyjNvsGy5uuL1xu01Px0L5vsClAvzDLYrjFsdtl7LZ/k4D8J10h7aRzsN5m2ZuL3hfgv3QbwepsRte6xNFsJjUw5rR1w+a5tibP22XdWOQ9w+a3clwuMT7yNbb7xvw/1ny4fbUW6bbXsslDsmzFfbtnB9lLV0O57VjqXl2zLhNtq6jHhZCLfR2muE20II2xru7zgvJmxHte3JwsrYdjGlbUa1fVypbqhWH1Am3DdGuB9sf0G87w3qCMsb1BPXT5m4HfG2ETfCum0/W77Fw3VYWUK8nixs3bY/y21juE/C/HLlw3bbMa6EnQ8EliXEx69cfcRJt/OvO22NzxPybf9CGA/L0p5w/zJPmmHHxkJYJ1ibCdQZLpuFtb3ScQrbFJ8bzIdtiLc7Jm6/hXCbIc639pWD9Vo77Jhb3NYZQhvD+lk+hPbEbYphH4R1WPjGN77hp1mEv2HxOmhDGLdyRri+uL3h9rciO/En2TFCiB6A5YAnfV1GIgssKlhP62X1qQWshliyZLkRovXgnoTrRH/+5jQSErVCdANeX/LKyPzt7DVQlj+XELwS7s+fWF4186q1kj+oEKJ54Z7UyvcjfVFMiG5Az1MsYYgVAr5PErQixnyJlyxZkqb0PfjaDRs2rGZ/QyFE89Hq9yNZaoUQQgghRO6RpVYIIYQQQuQeiVohhBBCCJF7JGqFEEIIIUTukagVQgghhBC5R6JWCCGEEELkHolaIYQQQgiReyRqhRBCCCFE7pGoFUIIIYQQuUeiVgghhBBC5J4BFbV8RnLp0qVpLBu+ZU45PjsZY58rXb16dZrSCemELFhnubyeUGt9Vi5ur21jVh2UJZ3vORtheULW9vcUPrUZrqsclGH/x9jnQaknhjTywmNOPeG2NAq17odKxMeJEB8r0spdA+zLcvs4K130jHoc6xg79vW8Ng2OfdbvoejEfjc5DvWE8yTrt6236JrupK/2RaXf2t7QnXOiL35rRCkNb6m95ppr3Lhx49zChQvTlFL41vmaNWvSWIFqJ+6sWbPSuf6Fb7KzLYsXL05TSmFb4ptgVlnKrVq1yvGFY74tP2rUqDSnf+BGsX79ej+fddOmfStWrEhjnSxYsCCdK8AFPnToUL8dhGnTpjXNDzvnYHicwmPVF0KnlelLAdmqcP6yT0X/0x1BrnO/sZBoHXgaXtQiZk2EZl24Y8eO7SJ4V65c6QVSFgNl4eDHp7293c2fP7+LuDPYlljEIg7jbUEgjRw50s+PGDHCT/vzR40HDdo6derUTNFtwjRsE/MIeoSewb4gGJMnT3YbNmyo6ce80Zk0aZIXsXacYOLEif5YlnuoEc2DPayFx79e8EA5Y8aMNCb6k+XLl5f8ZtULjqcZCkTfwPXIb3C96c45QTnKi76joUWtiSJuDOUE1OjRo/00FEKIRksPoQwCePbs2WlK/4EQRNBws0PcZVmTEXWhhdO2v62tzU+zWLt2rZ/2xc2zHOxD2nryySeXFegcr9CCzrEbM2ZMGqsM+6inIKixXBBCqy/7mzhP0ZYfP+BYOqE32LHN+gGNhfxAwH7AosB22mszs8xlbT9x9pXl2TLAvNVl+9usRxbih5tyeRDmhdeIveIL8+2atwelelnBw20lWJ22XWE+83E8LGvLMh+2Pzw3ody2WT2Wb+exrYf9Eq4/rjfe31YvEM9qE8vwUAakG+THZY14PSFh++K8Su2rJ7YfLbDdRtY+DMuH22rnITAN9x/BjjfE22bHDMLz2dZv67U3b5zXtj/C9VDeiM/9sE3W7krbDmHdcR7rsrxwvVDpvKsn8W9T+LtAPGwH7Q/Lh9tD3JalveF2E0LCOgl2XG1f2rLUZ8eSZbgfYryyfRWWJZ/5cD+G64n3b9i+eP/G+0QEJE8vAwarX7JkSRrrSiICO+bMmePnN2zY4MuHJBe0Xz4st2rVqo5ENPppXJ5ypLNMPTe9lvpoK+sGytNGw7aNaViObbIQljdYhlBpH3YX9lHWugzaRhsNyobrt7bG5Zi37SvXXpartO5q0HaCEe4bpllxg/XasnY8etoW9kG47ZWodPzK1dOd+rNg2XDbOFa0IzzvwvrJC/cV85QB9lmYB2G+1c0+BeZtPWx3uB7m42NgZWlvuJ6s42Vluwv12P6w9hphnq3H1mvnUByHuE3Mh/ucuB33eD9kbZvtT6CsxSkbrod43N5wPcQN5iu1KSxLe6xNEJaNt5Vytj1Z+zPetnLr7A22XtYBtMfWE+fZPjSYj+O2LGWt/UzDeojH+9P2SbxtlAuPeXjNxe0jL1423oe2HmtTSKVtD9cL5Nm5xTTcnrAe255y294bwjbZ9lVqr63Xti2O27LMW/upI6yHedtuW84It83aY2WBPDse8X6grOVBmB+fE7TBypIXto9l4vYxhXiftDoNbanlqQdrIJiFkyebGKyy5oJQziLIkxZPWf1p0TTsKc/WjQWPpzlLDwldENimww47zM9nkRw/H7CqZO2XvoC2YYU1pkyZkumjzLYmPwB+G207K1lgaT/7pDevZkIrqFk4tmzZ4qeGWU8HDx7sp5QjsG6sz0A7kx8XP18vwqfqRniyDq8RjinXlp2fXHN27IzkRzadcy75EXW33357GnN+WcMsIfZ6nDqTH1z/pgKYtzclHIvQN5t1nnfeeT5u17tdCxAeE/LtGNcT2ss1ZbCO+LWwnSd2Dlmbw3MqC64Vg/1g52a4H4D5uI5KvwPsFzt2XIucy8A+Zz12ztvUjhGUa1NMpWvL1mNtYH/Y+TNo0CCfZr9PYT21tK9esE+t/m3btvnp9u3b/RTCc4s2cY4blfYL56j9rvFGLTyOnEe2T2z58LjyW18L3AfC9nDMyr0hg/B6hErbTt3h7znrsXvpkCFDSu5TYT30D2Gf2bZzTVC23LnfU6if/Wjr2bRpkz+3QuwctnPN7kcWD49zSLjdWEKpG2r5Dah0PcZkvTUG24cGv4t2XPktYTvtWuC+aL+ptfxetzINK2rtYPKDYkKAi8YuuBAuNA4qJx8nRdYJx4lvP6b9DSch7YtFTXjDNswFwW7yduJWggsjFBl9Cft35syZxe1AUJe7oGgXLghsZ6UfcG541Bn+kPQEzhlrF+vj3KkF+9GzH8Hews2AfRLCthFCcVgN6ilHvV/3cW3Zvqtlv8U/8jFWFyHcFywXrotXbCHx9T4Q2PoJ/QG/W+E663FsTVxk/e70hErXFmIgbLMJEGB+1apVJb8ZoWitV/uqEb7KRUz0B7xOtnX29vc5/s3tDpW2nf0f/s6E89xXEbm4NtjyoWjlXmDp5jLRF3BuZa2/L7H1EfoKfgtD10J7KAbu+9wrONbWjvAe293f61aiYUUtFx8H1cQAgQuQkCWgeHLh4uUAx0KQC4Hl7IYZ+ov1x0XCxc/6w21h2+ypLIS2U5antvAprhHgZsR+DreDQFqWQDcLOiLdrFsx9RK0wBM6P8LUVU10hVR7ou8uZs0Ib949wZ7WY7hBhsKhHmQd10oPVNWEV1xX2LHJ0tg2roFY5ITL9feDKOcjvxO2/lr9wHvDJZdcUrL/63FsrY5wWyzY+dkdKl1bsYUy/k3lPLJ1243aytSrfZVgXZxniGvq749zinsUwsPO56y3Wd3B9n0YaqHatrP/Qyt0bJHmurX1cT8KjRPELc9CvX+X+G0IfxPCNwt9RX/9BvAbatZhMCu6wXVgbbCHC6O7v9etREOKWi5EfhCsZ7/BBVNOQHHisUyWRZDlwoNv1jLm630RxnBRcoHE66kkfNhGtqVcZ7fwqY0pP1r9cbHzoJF1gZd7Hcbx4weJkHXBse31ErQx9mNYC3Ze2WtxuxH0Brt5hw9gzHfHysI+49wJrZl2syz3kNAT7NWhtZV9Fz/whTdljlm5883Oa3vdbOcrdcbnrl0TiHfbVgSeQVmrpxJ9eQ2zrf1JT849ytuxCh+G7bWk/caw38P9X4nQahQTX1vx68/QHcHOJcPq5Zj1pn29ITzH+gv7bamF+I0Rr8nD85DfA3uo7O65H287dYdvP0N3BNYTdl7i4cXWa7/5dt5xnYbHua/o7cNBT+jOb0ClTt0x8X0zdEdgf4aGAyzoXFNQy+91KzPgojY0rxM4kGa5yLpgywkofiChnP9KNVh3b39Mw+0gcGJyEYa+OyGcwFmvwkwwxKIe2Cc8ddsrIaYIqFAkhxdDT+CCibeFJ3zSbT+HlBPotJXjSMjCfqDidfX0OHDj4AeIOmxYt1pf++GzhLWJZfnx4Mm4N7BPuGGHr+7sWMUiPusaMGgTlu6wDo5/PZ/KqYt2WVtpD+sIrz8eFq0N7Bs75lmw3XYcbF9SPj53rS7bFraVa9vyOH6hhbcSnGPU29trmPXRfmsD7SXelzcMfFDtmmM/cyyI1wr7if3M8mDWONvfdn7Z+VfLuWNlrM5K11Z8XClnlluOO8eYdGsDZaFa+xBV4QNdT2E9tNfaxz2C/RWPb15P2AbOSTsu9ltno9VUgvayHIHzjnOS9ts+5BoJLeOVzv1q207d4bXNvF1zdh5ZHsfV+jzYcbXtI4/rxCCtt9cisB5bB8Hul/Wouxy9+Q3A9ZFrNx7JIIv42gDb5/Fx4RqxkZE4tyr9XnP/iO/FrcROyQ22/mayHMJJYBajvMONIH7NJERP4YeTH1F+hEVjwc2Tm1mzXu/6LcsnzXQ/zRsIflwZWvX3uqFHP+hPeAJshguQE7o7r0CEEKIR4U1XPd1sRP/RLPfTPMJblVY2QMhSK4SoiCy1jUuzW2qFEKI7SNQKIYQQQojcI/cDIYQQQgiReyRqhRBCCCFE7pGoFUIIIYQQuUeiVgghhBBC5B6JWiGEEEIIkXskaoUQQgghRO6RqBVCCCGEELlHolYIIYQQQuQeiVohhBBCCJF7BkzUrl692n9+c+PGjWlKfeCzkWeeeWYaqx98h3z48OFd5mPYHraL7csj7Dv2YXfp6XKie3BuLV26NI31DM5dzmEhhBCimZClVogGBgGLkBVCCCFEZZpO1C5fvtzNnz8/jdWPGTNmuPXr16ex8gwdOtR1dHS4kSNHpilC1A/OrYkTJ6YxIYQQQhgNI2rttb2F0IWA19q8LrU8Xp+G5UNXgND9gCnByhFCtwBzgbAQvpKlTluWaZbLAeuyZc2NIsv9wMrE6fE2x6+VWZ/lVXtdHJYN28lytDPMj9dj6YRqxG3KcjmI91WWtdHqIITtsbKEsA6g3nC5kPhY2vGoBnWG54gR1hW2L15PfJzDNlJvSJgXb1t8LlAvYdKkST6fNJvSnngfA3HSIau+GMqGx8+WqXXfCSGEEA1FxwCxatWqDla/YcMGHx82bFjHkiVL/HycN27cOB83mI/jtixlp02b5ueZhvUQJ98gj3UBy4d10p6w7Jw5c3yazYfrtDiwrrBeliEfbB2Vttlg3XFbrWwM22XbDGFZa5u1J9wOCPeXtT1cb0i4/+KyYV68DttuI9wnVg/TrONudcb7J9xmq8O2OV5fJWz/2DqB9sV12/4L51lPuJ3khetl3rbT2mTrCfcXkGftD7c13pawHPPWFmunwbyt2+qzddv+t2UsnXrDNgkhhBB5omEstbzat9eq27Zt89Pt27f7KSQiIJ1zLrkpu+SmnMYK8S1btqSxUpKbtHcJgLa2thIXgmT7i24CtnxopRozZkw61xXWae3FNQFiaxjxRDC4k08+2ccpz3LXXHONj2NZmzVrlp+nHbQHaEN7e3sxD9j+RYsWpbFScLcwlwtrf7g/WKdt55AhQ3ybwNYzefJkH2c/hfs5ZsGCBW7KlCl+vlrZcsT7xNw1mC5evLjkeLH9tI92Dho0yKeZJTLcZvZneDxsGlpYK8Gytk5r33nnnefjpNMm2gaUnT17tp9nPbFLSiIM0znnz9Hbb7/dz3Ps2F+2Hva5bZu109odnguVoL41a9b4efaBHQ+rz85L6gvPO8O2be3atT6+cuXKiue8EEII0cg0jKgNXwGXE2/1JnQfMPFRK/GrXzAxbljchAyEy+H/C9aG+FX+qFGjinkIynIgYqzc2LFjvYCpBXtoMMFYC4MHD07nCg8JPSXcJwYCL0wP20X6qlWr3MyZM4vbGopWhKilE3oL+9DqQnwaiFjilhe7GMSEopdjaMtxbENqPWYho0ePdgsXLvTznL/2cGLYugj2IBPDQ4pdb7TPHjaEEEKIvNEQohYxww0V0YKFqi86esVgkUOccLNnnaFVtBZiCx2Egg8szvYZ8XLEWT+B9pglEmx/WDARHEPbsQpSJqtd5TDRGFrEqxEK902bNqVz3SfcJwbCNUyP22UWTAIWUfxNrTyi0PIsmOWzJ9h5YSE8Jy2NMpy3lSzC4UMMltSwToKJ+HKisxJsH8uxfo67WeONeF1muQ0ZMWKEP++og32Y9bAhhBBC5IGGHP3gkksuSef6D3ulXCsmJgAhiiCIRUX82jd+9Y4FLRRElMU9AGHBa+GwTYijalZBoL5aBVK8Hnu4KAeizKx6lcqai4MJztDynvUq3PZD+EoeQncE8iln2AMDeezP8HiwnynLtLtY+8JzkLo4xrQrrNcEYPgwEz4cYVU2dw2m7C/bNuqz7THxbe0P1xM/KMVwTFjn1KlT05TO+uwByeqz+kPsHOABIaxDCCGEyB0dA0TceSW5Ofs4gQ4ribAodnRJbrrFjjsQ5kG5skyJG5ShrEGerdPaY51w4nWEy9p8uLxtB1Orz7AycbqVtRBuI7AOywu3I4Y2h3WE2x1vs5UNCddD+UrrCsuG64n3NfNhnUxDLM/yjXBbwnaD1WMh3Jd2/CzYcQTaEq4jhPR4PRDWFR6XeD1hvVY2Kw/i9ts5A/G5ELbf0mw+zLP2hHVBXF/YFrY3jNs+D/cnZcL1CCGEEI3OTvxJbmhCdBuzHPeHu0hvwFKJZTjr9Xs9wRqaCMFeuT0MBFiEsSR3x3VFCCGEaDQa0v1ANCa4QISvtHmdTmelRgdXAnWAKg8jKMj1QAghRN6RpVbUDEJ22LDOXvrTpk1reCttf5JHSy1t5pjKSiuEECLvSNQKIYQQQojcI/cDIYQQQgiReyRqhRBCCCFE7pGoFUIIIYQQuUeiVgghhBBC5B6JWiGEEEIIkXskaoUQQgghRO6RqBVCCCGEELlHolYIIYQQQuQeiVohhBBCCJF7JGqFEEIIIUTukagVQgghhBC5R6JWCCGEEELkHolaIYQQQgiReyRqhRBCCCFE7pGoFUIIIYQQuUeiVgghhBBC5B6JWiGEEEIIkXOc+/977TDvblkrEQAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring RWA infestation, resistant wheat plants induce the upregulation of POD and β-1,3-glucanase activities throughout treatment to modify the cell wall by regulating callose and lignin accumulation (Forslund et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Miedes et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rajninec et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mafa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Other studies suggested that the β-1,3-glucanase regulates callose by degrading it into oligosaccharides referred to as DAMPs, which induce plant innate immune response (Forslund et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Luna et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rebaque et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is important to note that the majority of the studies reported in the literature used and characterised the apoplastic POD and β-1,3-glucanase (Forslund et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Mohase and van der Westhuizen, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Moloi and van der Westhuizen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mafa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, there is a lack of knowledge concerning the characterisation and physiological functions of cell wall-bound/associated POD and β-1,3-glucanase. Herein, the POD and β-1,3-glucanase associated with the cell wall were extracted from the cell wall region of RWASA2 infested susceptible Tugela, moderately resistant Tugela \u003cem\u003eDn1\u003c/em\u003e and resistant Tugela \u003cem\u003eDn5\u003c/em\u003e to study their substrate specificity and characterisation to understand their functions better.\u003c/p\u003e \u003cp\u003eThe results show that RWASA2 infestation induced significantly higher POD and β-1,3-glucanase activity levels in the cell wall regions of the moderately resistant and resistant wheat cultivars. β-1,3-glucanases were highly induced during wheat-RWA interactions (e.g. \u003cem\u003eDiuraphis noxia\u003c/em\u003e) or wheat-pathogen (e.g. \u003cem\u003eFusarium graminearum\u003c/em\u003e) interaction, which is associated with immune responses in wheat cultivars (Mohase and van der Westhuizen, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e; Van der Westhuizen et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Mohase and Taiwe, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Several studies used IWF extraction methods to collect the apoplastic fluid containing, among other proteins, β-1,3-glucanases and POD (van der Westhuizen et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Moloi and van der Westhuizen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mohase and Taiwe, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These enzymes were linked to resistant responses during RWA-wheat interactions because they were significantly induced in the resistant cultivars compared to control. For example, Van der Westhuizen et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) reported that apoplastic β-1,3-glucanase accumulated in the RWASA1-infested resistant Tugela \u003cem\u003eDn1\u003c/em\u003e played a significant role in the defence mechanism. However, the authors reported that the role of β-1,3-glucanase in defence against RWA was not clear (van der Westhuizen et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Other studies argued that β-1,3-glucanase regulates callose accumulation and mitigates the phloem and plasmodesmata channels clogging, allowing a more efficient cell-to-cell communication (Botha et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Pirselova and Matusikova, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Silva-Sanzana \u003cem\u003eet al\u003c/em\u003e., 2019; Walker, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To the best of our knowledge, the current study is the first to extract the cell wall-bound β-1,3-glucanase from the susceptible, moderately resistant and resistant wheat cultivars infested with RWASA2. The β-1,3-glucanase extracted from the moderately resistant Tugela \u003cem\u003eDn1\u003c/em\u003e and resistant Tugela \u003cem\u003eDn5\u003c/em\u003e hydrolysed the MLG and curdlan (β-1,3-glucan). These observations indicated that the cell wall extracted total protein contained β-1,3-glucanase which could hydrolyse the MLG or contained MLG specific glucanase enzyme. The β-1,3-glucan specific enzyme was possibly involved in callose regulation, while MLG specific enzyme modified the hemicellulose content of the resistant wheat cultivars infested with RWASA2.\u003c/p\u003e \u003cp\u003eBourdon et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) used wild-type and mutants of poplar plants to demonstrate that the callose deposition in the cell wall regions led to cellulose microfiber hydration, resulting in cellulose microfibril aggregates. These microfibril aggregations created gaps (mesopores with sizes ranging from 4 to 32 nm) between the cell wall polysaccharides and gaps between polysaccharides-lignin regions. Callose is semi-permeable, and its hygroscopic properties may impact the diffusion of monolignols, laccases and peroxidases necessary for subsequent lignin polymerization. Additionally, Bourdon et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) proposed model indicated that lignin content was reduced in the cell wall regions close to callose aggregates between the microfibrils. Mafa et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed that the resistant wheat cultivars infested with RWASA2 induced significantly higher apoplastic POD and β-1,3-glucanase activity levels, which were correlated to the cell wall reinforcement. The current study shows that in addition to the apoplastic enzymes, there are cell wall-bound POD and β-1,3-glucanase enzymes, which showed significantly higher activity levels upon RWASA2-wheat interactions.\u003c/p\u003e \u003cp\u003eThe significantly higher POD activity levels were also reported in previous studies (Verma et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Moloi and van der Westhuizen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mafa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). They reported a significant increase in POD activity, possibly involved in cell wall stiffening and strengthening by cross-linking the lignin content or lignin-hemicellulose connections. Ostergaard et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) proposed that POD activity in the cell wall is involved in the polymerisation of monolignols into lignin during cell wall lignification. In addition, Verma et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) observed that POD isozymes were abundant in the plant cell wall region and linked them to plant defensive responses. These observations support findings of the current study, which show the significantly higher activity of the POD extracted from the cell wall region of moderately resistant Tugela \u003cem\u003eDn1\u003c/em\u003e and resistant Tugela \u003cem\u003eDn5\u003c/em\u003e infested with RWASA2 for 14 days.\u003c/p\u003e \u003cp\u003eThe biochemical characterisation revealed that POD had optimum activity in the acidic pH range (pH5) for the Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e samples. However, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e extracted POD showed a broader pH optimum because they also showed optimum activity at pH 6. Peroxidase from Tugela \u003cem\u003eDn1\u003c/em\u003e leaves displayed an optimum pH of 5.6 (van der Westhuizen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1998b\u003c/span\u003e). In addition, soybean and lettuce stem POD displayed optimum activity at pH 5 (Hu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Feltrin et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Although the studies did not specify if they were cell wall or cytoplasmic POD, their optimum pH was comparable to the cell wall-bound POD. All samples lost more than 70% of enzyme activity in the alkaline pH range. Other studies showed that cytoplasmic POD had an optimum activity at pH 7 (neutral range) in wheat and tobacco samples (Bania and Mahanta, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hamid et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This observation validated that the POD from the three wheat cultivars infested with RWASA2 were extracted from the cell wall region, which is generally acidic.\u003c/p\u003e \u003cp\u003eIn addition, β-1,3-glucanase extracted from RWASA2-infested Tugela, Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e displayed optimal activity at pH 5 and retained 90% relative activity at pH 4. Taiz and Jones (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1970\u003c/span\u003e) demonstrated that β-1,3-glucanase on barley seed displayed optimal activity at an acidic pH range between 4.47 to 5.5 using laminarin as a substrate. Wheat apoplastic β-1,3-glucanase displayed optimum activity at pH 6.5 (van der Westhuizen et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our findings showed that the β-1,3-glucanase extracted from the three wheat cultivars were cell wall related because they showed optimum activity at pH5 (and retained most activity at the acidic pH range) during curdlan and MLG substrates hydrolysis.\u003c/p\u003e \u003cp\u003eThe temperature studies displayed a gradual increase in POD activity, reaching optimum at 50\u0026deg;C in the three wheat cultivars. The optimum temperature profile was similar to the POD extracted from wheat (Hamid et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Altin et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The β-1,3-glucanase optimum temperature was investigated in both the β-1,3-glucan and β-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-glucan substrates. It displayed maximum enzyme activity at 40\u0026deg;C when hydrolysing curdlan and 25\u0026deg;C during the hydrolysis of MLG. The β-1,3-glucanase retained more than 80% relative activity at 40 and 50\u0026deg;C during the hydrolysis of MLG substrate. However, Zhang et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported β-1,3-glucanase displaying maximum activity at 50\u0026deg;C. In addition, the β-1,3-glucanase sourced from tomato roots displays optimum temperature at 40\u0026deg;C (Mohamed et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These findings confirm the observation that the protein extracts contained two isozymes or β-1,3-glucanase and MLG specific enzymes. The thermostability assays for β-1,3-glucanase also validated our claim, showing that Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e extracted enzymes were thermostable (retaining more than 70% relative activity at 70\u0026deg;C). Future research should elucidate the function of the MLG specific β-1,3-glucanase or β-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-glucanase enzymes bound to the cell wall region.\u003c/p\u003e \u003cp\u003eβ-1,3-glucanase mechanism of action was assessed with mixed-linked glucan, curdlan and Azo-CMC substrates, representing β-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-, β-1,3- and β-1,4-glycosidic-linkages, respectively. The enzymes showed a higher activity towards a substrate that was constituted by β-1,3-glycosidic bonds compared to β-1,4-glycosidic bonds or mixed linked β-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-glycosydic bonds because it displayed higher activity on curdlan, followed by MLG and CMC substrates. Rodriguez-Mendoza \u003cem\u003eet al\u003c/em\u003e. (2019) studied β-1,3-glucanase displaying higher affinity and activity towards β-1,3-linkages of laminarin and curdlan. β-1,3-glucanase showed higher activity on laminarin and curdlan because they are chemically similar to callose, which is made up of glucopyranosyl units joined by β-1,3-glycosidic bonds (Moravcikova et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mafa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Walker, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, a few studies have investigated the preference of β-1,3-glucanases activity toward substrates made of different β-1,4-glycosidic-linkage, β-1,3-glycosidic-linkage or β-1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,4-glycosidic linkages. Because the β-1,3-glucanase from RWASA2 infested moderately resistant and resistant Tugela cultivars was significantly highly active compared to controls and susceptible cultivars, we investigated the mode of action of this enzyme using LAM5 to LAM2 as substrates. Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e extracted β-1,3-glucanase were significantly effective on oligosaccharides with longer DP according to the TLC and LC-MS results. The susceptible cultivar had the lowest activity on all laminarin-oligosaccharides. Driskill et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) corroborated our findings that the β-1,3-glucanase displayed the highest hydrolytic activity on oligosaccharides with higher DP (e.g. LAM3 to LAM6). In addition, a β-glucanase with broad glucan substrate specificity sourced from a fungus (\u003cem\u003ePodospora anserine\u003c/em\u003e) showed the highest hydrolytic activity on laminarin, followed by LAM3 to LAM6, in that order (Lafond et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePrevious studies have demonstrated that the RWA-wheat interaction led to upregulation of defence-related proteins in wheat plants. Among these proteins, the apoplastic and cytoplasmic POD and β-1,3-glucanase were demonstrated to play a physiological function of modulating ROS levels and regulating callose accumulation in the infested resistant wheat cultivars, respectively. Herein, we demonstrated that cell wall-bound POD extracted from RWASA2-infested moderately resistant Tugela \u003cem\u003eDn1\u003c/em\u003e and resistant Tugela \u003cem\u003eDn5\u003c/em\u003e cultivars had significantly higher activity compared to the control. In contrast, this enzyme displayed significantly reduced activity over time in the susceptible cultivars. This finding confirmed that the cell wall-bound POD has a defensive role against RWA infestation in the resistant wheat cultivars. In addition, two β-glucanase enzymes displayed significantly higher activity in resistant cultivars than controls during RWASA2 infestation. To our knowledge, this is the first study that links MLG specific cell wall bound β-glucanase to wheat defence responses upon RWA infestation. The characterisation confirmed that the enzymes studied were cell wall-bound because they had optimum pH at the acid range (pH). The temperature and thermo-stability assays showed that POD and β-1,3-glucanase had similar profiles, displaying optimum temperature at 40 and 50\u0026deg;C, respectively. Also, both enzymes were thermophilic, losing more than 90% activity at 70\u0026deg;C. In contrast, the MLG specific β-glucanase displayed temperature optimum at 25\u0026deg;C but retained more than 80% activity at 40\u0026deg;C in Tugela \u003cem\u003eDn1\u003c/em\u003e and Tugela \u003cem\u003eDn5\u003c/em\u003e, confirming that this enzyme was different from β-1,3-glucanase. In addition, the mechanism of action analysis confirmed that the MLG specific β-glucanase and β-1,3-glucanase were associated with the wheat defence responses against RWASA2 infestations because their activity levels significantly increased in the resistant Tugela \u003cem\u003eDn5\u003c/em\u003e. In conclusion, the characterisation findings suggest that POD and β-1,3-glucanase function under the same conditions, suggesting they synergistically act to reinforce the cell wall during the RWASA2 infestation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. Mafa received funding from the NRF-Thuthuka (Reference Number: TTK2204102938). S.N. Zondo received the National Research Foundation Postgraduate Scholarship for his MSc degree.\u0026nbsp;Authors are grateful to\u0026nbsp;Agricultural Research Council – Small Grain (ARC-SG) Institute for providing the seeds used in this study. Any opinion, findings, and recommendations expressed in this material are those of the author(s), and therefore, the funders do not accept any liability in regard thereto.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. Mafa, and S.N. Zondo conceived and designed research. M. Mafa sourced funds and supervised the study. S.N. Zondo conducted experiments and wrote the wrote first draft. M. Mafa, S.N. Zondo, L. Mohase and V. Tolmay analysed and interpreted data. L. Mohase and V. Tolmay Co-supervised the study. S.N. Zondo and Mafa wrote the first draft of the manuscript. All authors read, edited and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental datasets used in the current study is available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations Conflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAltin, S., Tohma, H., G\u0026uuml;l\u0026ccedil;in, I., and K\u0026ouml;ksal, E., 2017. 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Peroxidases, phenylalanine ammonia-lyase and lignification in peduncles of rose flowers. \u003cem\u003ePlant physiology and biochemistry (Paris)\u003c/em\u003e. 29(2),147\u0026ndash;151.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"β-1,3-glucanase, Peroxidase, wheat, Russian wheat aphid, wheat-pest interaction, cell wall bound enzymes","lastPublishedDoi":"10.21203/rs.3.rs-3968206/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3968206/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWheat plants infested by Russian wheat aphids (RWA) induce a cascade of defence responses, which include increased activity of β-1,3-glucanase and peroxidase (POD). There is a lack of information regarding β-1,3-glucanase and POD synergistic effects on the plant cell wall modification and characterisation during wheat-RWA infestation. This study aimed to characterise the physicochemical properties of the cell wall-bound POD and β-1,3-glucanase during RWA-wheat interaction. The susceptible Tugela, moderately resistant Tugela \u003cem\u003eDn1\u003c/em\u003e, and resistant Tugela \u003cem\u003eDn5\u003c/em\u003e cultivars were planted in a glasshouse to a seedling stage before being infested with RWASA2 for 14 days. The findings showed a significant increase in β-1,3-glucanase and POD activities in the infested Tugela \u003cem\u003eDn5 \u003c/em\u003eand Tugela-\u003cem\u003eDn1\u003c/em\u003e cultivars over the 14 days. However, in the Tugela enzymes were repressed. In addition, it was shown for the first time that β-1,3-1,4-glucanase activity specific toward mixed-linked glucan was significant in the resistant cultivar over 14 days. β-1,3-glucanase, β-1,3-1,4-glucanase and POD displayed optimum at pH 5. β-1,3-glucanase and POD displayed temperature optimum at 40 and 50°C, respectively. However, β-1,3-1,4-glucanase had temperature optimum at 25°C. β-1,3-glucanase and POD had a thermo-stability at 37°C followed by about 80% relative activity at 70°C, but β-1,3-1,4-glucanase displayed thermostability at 25°C and retained more than 75% at 70°C, confirming that β-1,3-1,4-glucanase and β-1,3-glucanase induced in the resistant cultivars cell wall were two different enzymes. Mechanism of actions and oligosaccharide displayed that β-1,3-glucanase was highly active against β-1,3-glucan and required a triose and higher oligosaccharide to be active. Our findings demonstrated cell-wall bound POD and β-1,3-glucanase activities significantly increased in wheat after RWASA2 infestation, revealing they acted synergistically to reinforce the cell wall to deter RWASA2 feeding in resistant cultivars.\u003c/p\u003e","manuscriptTitle":"Functional characterization of cell wall-associated β-glucanases and peroxidase induced during wheat-Diuraphis noxia inteactions.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-06 11:32:51","doi":"10.21203/rs.3.rs-3968206/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-04-12T03:39:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-04T08:44:45+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-01T08:22:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-26T02:44:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2024-02-22T04:53:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"37f87851-156e-402a-a00f-0a6aafb3fc5e","owner":[],"postedDate":"March 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:10:50+00:00","versionOfRecord":{"articleIdentity":"rs-3968206","link":"https://doi.org/10.1007/s11756-024-01734-1","journal":{"identity":"biologia","isVorOnly":false,"title":"Biologia"},"publishedOn":"2024-07-22 16:16:03","publishedOnDateReadable":"July 22nd, 2024"},"versionCreatedAt":"2024-03-06 11:32:51","video":"","vorDoi":"10.1007/s11756-024-01734-1","vorDoiUrl":"https://doi.org/10.1007/s11756-024-01734-1","workflowStages":[]},"version":"v1","identity":"rs-3968206","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3968206","identity":"rs-3968206","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}