Trichoderma strains and chemicals
Trichoderma isolates used in this study was obtained from Prof. Lo’s lab in Department of Biotechnology at National Formosa University, Taiwan. The isolates were maintained and sporulated on potato dextrose agar plates at 28 °C for 7 days. Chitin from the crab shells, chitosan (DA 85%), carboxymethylcellulose (CMC), starch, 3,5-dinitrosalicylic acid (DNS), p-nitrophenyl, p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG), GlcNAc, and 4-MU-α-GlcNAc3 were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). N, N’-diacetylchitibiose was from Toronto Research Chemicals (Toronto, ON. Canada).
Preparation of colloidal chitin and glycol chitin
20 gof powder crab chitin was mixed with 100 ml of 50% H2SO4 at room temperature for 2 h, followed by washing with water until pH 6.5–7.0. The suspension was passed through a 0.053 mm mesh sieve (Der Shuenn, Taiwan) to remove large particles. Afterward, the suspension was centrifuged at 6000 rpm for 10 min at 4 °C. The pellet containing colloidal chitin was recovered and stored at 4 °C until use. Glycol chitin (EG-chitin) was prepared using the method (Yamada and Imoto 1981).
Production and purification of chitinase
T. virens strain mango (105 cfu/ml of spores) was cultured in a chitin-containing medium (one liter contained 15 g of colloidal chitin, 0.7 g of K2PO4, 0.5 g of KH2PO4, 0.5 g of MgSO4·7H2O, 18 mg of FeSO4·7H2O, 1.8 mg of ZnSO4·7H2O), and incubated at 28 °C with shaking for indicated days. Trichoderma filtrate was collected followed by precipitation with 80% ammonium sulfate. After centrifugation, the protein precipitate was dissolved in 10 mM Tris–HCl buffer at pH 7, and dialyzed against the same buffer using cut-off 6–8 kDa dialysis membrane (Spectra/Por®) at 4 °C overnight. Then, the supernatant was applied to a chitin-bead affinity column (Biolabs). After washing out the unbound protein with 10 mM Tris–HCl at pH 7.5, chitinase was eluted with 10% acetic acid buffer. The collected chitinase was dialyzed against 10 mM Tris–HCl at pH 7.5. The activity assay was subsequently performed. Otherwise, it was stored at − 20 °C until use.
Identification of protein by MALDI/MS
Protein band in SDS-PAGE gel was manually excised and ground into pieces. After washed with 50% acetonitrile and 50% acetonitrile/25 mM ammonium bicarbonate, the protein was in-gel reduced and alkylated in 25 mM ammonium bicarbonate buffer containing 10 mM dithiothreitol and 55 mM iodoacetamide. Then, the protein was digested at 37 °C overnight by 0.1 mg of porcine trypsin (Promega, Madison, WI, USA). The tryptic peptides were subsequently extracted from the gel by 50% acetonitrile/5% formic acid, followed by MALDI/MS analysis using a quadrupole-time-of-flight (Q-TOP) mass spectrometer (Micromass Q-T of Ultima, Manchester, UK) in the proteomics Research Core Laboratory at National Cheng-Kung University, Taiwan.
Enzyme activity assay
NAGase activity was usually performed by using pNP-NAG as the substrate. 10 μl of protein sample was mixed with 50 μl of 50 mM phosphate buffer at pH 5, containing 300 µg/ml pNP-NAG. After incubation at 65 °C for 30 min, 50 μl of 0.4 M Na2CO3 was added to stop the reaction. The absorbance of the mixture was measured at 405 nm to determine the amount of p-nitrophenol released according to a standard curve of p-nitrophenol. One unit of NAGase activity corresponded to the amount of enzyme required to produce 1 µmol of p-nitrophenol min−1. For substrate specificity, 1.5% of various substrates including chitin, EG-chitin or CMC were used. After incubation at 40 °C for 24 h, the release reducing sugars were quantified by the DNS method (Ghose 1987).
The fluorometric assays were performed to determine endochitinase activity using a 4-methylumbelliferyl-ß-D-N, N’, N’’-triacetyl chitotriose (Sigma) as subtracts. Following the reaction at 37 °C for 1 h, the released 4-methylumbelliferone (4-MU) was estimated by a spectrofluorometer (Beckman, Fullerton, USA) at an excitation of 360 nm and an emission of 465 nm.
TLC and HPLC analysis of hydrolytic products
The purified NAGase (50 mU) was incubated in 200 µl of 50 mM phosphate buffer (pH 5) containing 1.5% colloid chitin. Then, the hydrolytic products were analyzed by TLC and HPLC. Using a solvent system, butanol-acetic acid–water (2:1:1, v/v/v), the aliquots of hydrolytic products were spotted onto a TLC silica gel plate (Merck, Damstadt, Germany). The plates were sprayed with solution, containing 1% KOH, 2.5% acetone, 4% ethanol in butanol, followed by heating in an oven at 100 °C for 5 min. Afterward, the plates were sprayed with solution containing 0.4% (w/v) dimethylamino benzaldehyde, 12.5% ethanol, 12.5% HCl and 75% butanol, heating in an oven at 100 °C for 5 min. The hydrolytic products were also subjected to HPLC analysis using a PolySep-GFC-P 2000 column (Phenomenex, USA) with running solution, acetonitrile: water (3:2) at 0.8 ml/min of flow rate under OD230 detection using commercial GlcNAc for comparison.
Antifungal activity assay
To obtain sclerotial bodies, Sclerotium rolfsii was cultured on potato dextrose agar for 2–3 weeks. Two pieces of sclerotial bodies from S. rolfsii was inoculated into 1 ml potato dextrose broth with or without the purified NAGase. Six pieces of sclerotial bodies were used for each treatment. After incubation at 28 °C for 24–36 h with shaking, the sclerotial bodies were moved to the plate. The hyphal growth inhibition by the purified protein was observed and photographed. The mycelium length was recorded.
RNA isolation, PCR cloning and RT-PCR analysis
The harvested mycelia of T. virens strain mango was frozen with liquid nitrogen, and subsequently ground into a fine powder. For total RNA isolation, 0.1 g of powder sample was mixed with 1 ml of TRIzol reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions. The mixture was stand at room temperature for 5 min, followed by mixing with 200 µl of chloroform. After centrifugation, the aqueous phase was recovered. RNA was precipitated with two volume of ethanol (> 99.8%), rinsed with 70% ethanol and dried on air. Finally, RNA was dissolved in 40 µl of water pretreated with DEPC.
The first strand cDNA was synthesized using SuperScript™ III reverse transcriptase (Invitrogen, CA, USA), and was used as templates for the following PCR cloning of TvmNAG2 or RT-PCR analysis. Based on DNA sequence of nag2 from T. virens Gv29-8 (TvNag2, accession number, XM_014099474), the degenerate primers were designed (forward primer dpNAG2-F, 5’-CTG TGG CCC GTG CCG ANN-3’; reverse primer dpNAG2-R, 5’- TCA GTA ATT CCC TGA CTC ACN-3’). After cloning and sequence analyses, the DNA fragment coding for TvmNAG2 without signal peptide was obtained.
For RT-PCR analysis of TvmNAG2, conserved degenerate primer TvNAG-midF, 5- GCG ACC CGA CCA AGA ACT GNN -3’; and reverse primer 5’-TCA GTA ATT CCC TGA CTC ACC G-3’ were used. For RT-PCR analysis of nag1, conserved degenerate primer TvNAG-midF; and reverse primer, 5’-TTA GGT GAA CAG CGT GCA AGN-3′ were used. Both DNA fragments (~ 350 bp) was separately subcloned into pGEM-T vector, followed by sequencing to confirm they belonged to TvmNAG2 and nag1. The primers for actin, 5’-ATGTGCAAGGCCGGTTC-3’ and 5’-GTCTCGAAGACGATCTGG-3’ were used and the expected PCR product was around 350 bp as well.
The similarity searches were accomplished via BLAST network at NCBI. The alignment of selected sequences was performed with CLUSTAL O (1.2.4) multiple sequence alignment at EMBL-EBI, and then modified.
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