Galacturonic acid

Total protein extracts were obtained by homogenizing uninfected and infected Arabidopsis leaves in the presence of 1 M NaCl, 12.5 mM Citric Acid, 50 mM Na₂HPO₄, 0.02% Sodium Azide, protease inhibitor 1:100 v/v (P8849, Sigma), pH 6.5 (2 mL of extraction buffer per g of tissue). The homogenates were shaken for 1.30 h at 4°C, centrifuged at 15.000 × g for 15 min, and the supernatant collected. Protein concentration was determined in the supernatants using Bradford protein assay method (Bradford reagent, Sigma-Aldrich) and bovine serum albumin as standard (Bradford, 1976 (link)). After separation by sodium dodecyl sulfate–polyacrylamide gene electrophoresis (SDS-PAGE; Biorad), proteins were analyzed by Coomassie blue staining (SimplyBlue™ SafeStain, Invitrogen). PECTOPLATE was prepared with 0.1% (w/v) of apple pectin (molecular weight range 30,000–100,000 Da; 70–75% esterification; 76282, Sigma-Aldrich, St. Louis), 1% (w/v) SeaKem^® LE agarose (Lonza, Basel, Switzerland, Catalog no: 50004E), 12.5 mM citric acid and 50 mM Na₂HPO₄, pH 6.5. The gel was cast into 120 mm square petri dishes (50 mL per plate) and allowed to polymerize at room temperature. Wells with a diameter of 4 mm were obtained with a steel cork borer and equal amounts of protein samples (2 μg of total protein in 20 μL) were loaded in each well. Plates were incubated at 30°C for 16 h, and stained with 0.05% (w/v) RR (R2751; Sigma-Aldrich, St. Louis) for 30 min. The plates were de-stained by several washes with water and the area of the fuchsia-stained haloes, resulting from de-methylesterification of pectin and the area of inner unstained haloes, resulting from the hydrolysis of pectin in the gel, were measured with Image J software (Abramoff et al., 2004 ). Pectinase activity of B. cinerea was obtained by culturing the fungus in liquid Czapek Dox medium (2 g L^–1 NaNO₃, 1.0 g L^–1 K₂HPO₄, 0.5 g L^–1 MgSO₄, 0.5 g L^–1 KCl, 0.01 g L^–1 FeSO₄) containing 0.5% Polygalacturonic Acid (w/v; P3850, Sigma-Aldrich, St. Louis) as the sole carbon source. The flasks were inoculated with 1 mL of conidia (4 × 10⁵ conidia mL^-1) and incubated on a rotary shaker at 100 rpm at 23°C for 3 days. B. cinerea PME activity was induced in the B. cinerea culture by adding 0.5% apple pectin (w/v; 76282, Sigma-Aldrich, St. Louis) and after 10 h the culture filtrate was collected. Recombinant AtPMEI-1 expressed in Pichia pastoris and purified to homogeneity (Raiola et al., 2004 (link)) was pre-incubated for 15 min with protein extracts before loading the mixture in the wells. Known amounts of commercially available PME from orange peel (P5400; Sigma-Aldrich, St. Louis) and of a Polygalacturonase (PG) from Aspergillus japonicus (P3304; Sigma-Aldrich, St. Louis) were used in PECTOPLATE. One PME unit is defined as the amount of enzyme required to release 1.0 microequivalent of acid from pectin per min. One PG units is defined as the amount of enzyme required to release 1.0 μmol of reducing sugar measured as D-Galacturonic acid from Polygalacturonic Acid per min. The area of the PME and PG haloes measured as above described were used to generate two standard curves, which was used to calculate the total PME and pectinase activity of the sample extracts.

The A. niger strains used in this study are listed in Table 2. Strains were grown at 30 °C on minimal medium (MM) or complete medium (CM) [51 (link)] either or not containing 1.5% agar. Liquid cultures were grown on a rotary shaker at 250 rpm. Pre-cultures for RNA isolation were grown for 16 h in 1 L Erlenmeyer flasks that contained 250 ml CM supplemented with 2% D-fructose. Mycelium was washed with MM and 1 g (wet weight) aliquots were transferred for 2 h to 250 ml Erlenmeyer flasks containing 50 ml MM supplemented with 25 mM mono- or disaccharide or ferulic acid, or mixture of 25 mM L-rhamnose and 25 mM D-galacturonic acid, or 1% polysaccharide or complex plant biomass (Table 3). The only exceptions were D-maltose cultures of N402 and ∆amyR strains that were incubated for 4 h and for which 1% maltose was used. These data originate from a different study [8 (link)], but were included to help with the grouping of the genes and assess the AmyR effect. Mycelium was harvested by vacuum filtration, dried between towels and frozen in liquid nitrogen. While N402 liquid cultures were performed on all carbon sources listed in Table 3 as well as on the mixture of L-rhamnose and D-galacturonic acid, the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX were grown on D-xylose, L-arabinose, maltose, L-rhamnose and D-galactose, respectively, and L-rhamnose and D-galacturonic acid. All cultures were performed as biological duplicates.

Esterase-mediated uronic acid formation was monitored continuously using the K-URONIC kit (Megazyme, Ireland). Kinetic measurements were performed in 96-well plates using a FLUOstar Omega (BMG LABTECH, Germany) in 200 μL reactions containing 50 mM sodium phosphate, 2 μL uronate dehydrogenase, and 16 μL NAD⁺. The buffer pH was at or close to the enzymes’ respective pH optima, due to substrate instability at higher pH, and where > 75% of maximal enzyme activity is maintained: pH 7.5 for O. terrae and S. usitatus enzymes and pH 6.5 for S. linguale enzymes. The substrates BnzGlcA, AllylGlcA, MeGlcA, and MeGalA (Additional file 1: Figure S2) (Carbosynth, UK) were dissolved in 100% dimethyl sulfoxide (DMSO); all reactions contained ≤ 10% DMSO. Kinetic assays were performed at least in duplicate at 25 °C using enough enzyme to ensure ≥ 2-fold change in substrate turnover versus auto-hydrolysis rates. pH-dependency profiles were generated with 2 mM BnzGlcA in a three-component buffer containing 25 mM acetic acid, 25 mM 2-(N-morpholino)ethanesulfonic acid, and 50 mM Tris–HCl, covering pH 4.5–9.5 [36 (link)].
Acetyl esterase activity was assayed using 4-nitrophenyl acetate (pNP-Ac; Sigma Aldrich) and 1,2,3,4-tetra-O-acetyl-β-d-xylopyranose (TetAcXyl; Carbosynth) (Additional file 1: Figure S2). pNP release was detected at λ₄₀₅ and quantified using an extinction coefficient 18.7 mM⁻¹cm⁻¹. Acetate release from TetAcXyl were measured using the K-ACET kit (Megazyme). Pectin methyl esterase activity was assayed with poly-d-galacturonic acid methyl ester (Carbosynth) and citrus peel pectin (Sigma Aldrich) in reactions containing 0.2% (w/v) pectin and 1 mg/mL CE15 enzyme. Reactions were collected at 30 min and 24 h, filtered through a 10 kDa Amicon spin filter, and methanol release through NAD⁺ reduction using alcohol oxidase (Pichia pastoris, Sigma Aldrich) and formaldehyde dehydrogenase (Pseudomonas sp., Sigma) as previously described [37 (link)]. Nonlinear data were fitted to the Michaelis–Menten equation using GraphPad Prism (GraphPad, US). In nonsaturable cases, k_cat/K_m values were determined by linear regression.

The Gal A content in hawthorn pectin was determined using the carbazole-sulfuric acid method according to Chen [21 ]. In total, 0.5 mL of D-Gal A standard solution (20, 40, 60, 80, 100 mg/L, respectively) was placed in a stoppered test tube. Then, 3 mL of borax sulfuric acid solution (5 mg/mL) was added under ice-water bath conditions and mixed with a vortex mixer. The resulting mixture was kept in a boiling water bath for 5 min, then immediately cooled. After that, a 0.1 mL aliquot of the carbazole solution (0.1%) was added to the mixture, boiled for 5 min, cooled to room temperature, and the absorbance was measured at 530 nm. A total of 0.5 mL of sample solution was used instead of the standard solution and the absorbance value was measured.