Characterization of a class III chitinase from Vitis vinifera cv. Koshu

JOURNALOF BIOSCIENCEAND BIOENGINEERING Vol.

95, No. 6,645-647.2003

Characterization of a Class III Chitinase from Yitis vinifera cv. Koshu AKIHIKO

ANO,’ TSUTOMU TAKAYANAGI,‘* TAKAYUKI TOHRU OKUDA,’ AND KOKI YOKOTSUKA’

UCHIBORI,’

The Institute of Enology and Kticulture, Yamanashi University, 1-13-1 Kitashin, Kojii 400-0005, Japan’ Received29 November 2002iAccepted 25 February 2003

A chitinase gene (Chi3K) was cloned from the genomic DNA of vitis vinifera cv. Koshu. The structural gene comprised 891 bp without introns and encoded 297 amino acids. The Chi3K product showed high similarity to the class III chitinase of K vinifera cv. Pinot noir. Chi3K was expressed using a bacterial expression vector for purification and enzymatic characterization of its gene product. The recombinant chitinase exhibited hydrolytic activity toward glycol chitin and its optimum pH was 4.0. It also inhibited the growth of Botrytis cinerea, which causes grey mold disease in grapes. [Key words: Vitis vinifera, Koshu, chitinase, antifungal activity] Plant chitinases are thought to be closely related to plants’ defense system against phytopathogens, because it has been demonstrated that plant chitinases are induced by fungal attack (1) or contact with some elicitors (2), and directly inhibit fungal growth in vitro (3). We investigated chitinase activity in Koshu grapes (Wis vinzjkra cv.) (4). Acidic chitinase activity (maximal activity at pH 4.0) in the grapes markedly increased after veraison (onset of grape ripening), and its induction by treatment with an elicitor (glycol chitin) was also observed after veraison. Several chitinase cDNAs were cloned from grape leaves and berries (5-7). However, the direct effect of these chitinases on phytopathogens and their enzymological characteristics remain unclear. We describe herein the cloning and sequencing of a class III chitinase gene from Koshu grapes. The cloned chitinase gene was expressed by recombinant bacteria, and the antifungal activity and enzymological characteristics of the expressed chitinase were investigated. Genomic DNA was extracted from Koshu grape leaves according to a modified CTAB procedure (8). The chitinase gene was amplified by PCR using two primers (sense primer, 5’-TCCCCCGGGACATACACGTTATTCAAG-3’ and antisense primer 5’-TGGTCGACCATTCATCGAGGA TGAAAGC-3’). The sense primer was constructed with reference to the cDNA sequence of a class III chitinase of I! vinifera cv. Pinot noir (5) and the antisense primer was designed from the promoter region of a Koshu class III chitinase gene isolated by genome walking (data not shown). Recognition sites for SmaI and Sal1 were attached to the 5’-ends of the sense and antisense primers, respectively. The

PCR product was completely digested with SmaI and SaA, cloned into pUC 18, and sequenced by an ALF express DNA sequencer (Amersham Biosciences, Piscataway, NJ, USA). The cloned DNA fragment had one open reading frame of 89 1 nucleotides. The deduced polypeptide of the DNA fragment had 297 amino acids with a size of 3 1.9 kDa. The amino acid sequence encoded by the DNA fragment showed 97.0% identity to that of the class III chitinase of Pinot noir and had conserved amino acids in the catalytic domains of family 18 chitinases (Fig. 1) (9). The cloned chitinase gene, Chi3K (DDBJ accession no. AB 105374), was excised from pUC 18-Chi3K with SaZI and SmaI and cloned into the polylinker site of the bacterial expression vector, pGEX4T3 (Amersham Biosciences). Escherichia coli JM109 was transformed with pGEX4T3-Chi3K (10). The expression of GST-Chi3K in E. coli was induced following incubation with 0.2 mM isopropylthio-P-D-galactoside at 20°C for 4 h. The induced cells collected by centrifugation were incubated with PBS (pH 7.4) containing

* Corresponding author. e-mail: [email protected] phone: +81-(0)552-20-8607 fax: +81-(0)552-20-8658 Abbreviations: CTAB, cetyltrimethylammonium bromide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfateipolyacrylamide gel electrophoresis.

MARTPQSTPLLISLSVLALLQTSYAGGIAI

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YWGQNGNEGTLTQTCNTGKYSYVNIAFLNK

60

FGNGQTPEINLAGHCNPASNGCTSVSTGIR

90

NCQNRGIfjVML~IG~GSYSLSSSNDAQN

120

VANYLWNNFLGGQSSSRPLGDAVL~I~F~

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INLGSTLHWDDLARALSGFSKRGRKVYLTA

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PQCQY5SGNTNNLLNSWNRWTSSINSQIFt.i

240

GLPASSAAAGSGFIPANVLTSQILPVIKRS

270

PKYGGVMLWSKYYDDQSGYSSSIKSSV

297

FIG. I. Amino acid sequence deduced from the nucleotide sequence of the class III chitinase gene cloned from Koshu genomic DNA. The conserved amino acids in the catalytic domains of family 18chitinases (9) are boxed. 645

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FIG. 2. Effects of pH (A) and temperature (B) on the activity and stability of the recombinant chitinase. The enzyme activity at various pHs (solid circles) was measured under standard assay conditions except for the use of 0.2 M McIlvaine buffer (pH 2.5 to 8.0). To determine enzyme stability at various pHs (open circles) each enzyme solution was mixed with 0.2 M McIlvaine buffer (pH 2.5 to 8.0) and kept at 4’C for 24 h. The residual activity of the treated enzyme was assayed after adjustment to pH 4.0 by adding 0.2 M acetate buffer (pH 4.0). Enzyme activity at various temperatures (20°C to 7O’C) (solid squares) was measured at pH4.0. Regarding the heat stability of the enzyme (open squares), enzyme solutions (pH 4.0) were pre-incubated at various temperatures (20°C to 70°C) for 1 h and then cooled in an icewater bath. The residual activity of the treated enzyme was assayed under standard assay conditions. Error bars represent SE (n=2).

1% Triton X-100, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.1% /3-mercaptoethanol, 5 mM benzamidine and 10 pg.ml-’ lysozyme for 5 min at 25°C and then lysed by sonication. The GST-Chi3K fusion protein was separated from the lysed solution by affmity chromatography with a Glutathione-Sepharose 4B column (Amersham Biosciences). After cleaving a GST-tag in the GST-Chi3K (fusion protein) with thrombin protease, the recombinant chitinase of Chi3K was purified by eluting the digested solution through columns of HiTrap Benzamidine FF (Amersham Biosciences) and Glutathione-Sepharose 4B. The purified recombinant chitinase showed a single protein band upon SDS-PAGE. The molecular mass of the recombinant chitinase was determined to be 34.5 kDa by comparison of its relative mobility upon SDS-PAGE with those of standard proteins (LMW Marker Kit; Amersham Biosciences). This value agreed with the molecular mass calculated from the deduced polypeptide including the thrombin recognition site. Chitinase activity was deter-

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FIG. 3. Inhibition of hyphal growth and spore germination of B. cinerea RIFY 4115 by the recombinant chitinase. (A) The result of the PDA plate test. After the diameter of the fungal colony at the center of the plate had reached 2.0 cm, sterile filter paper discs were placed on the agar, and 100 ul of the following preparations were pipetted onto the discs: 1, water; 2, PBS (pH 7.4); 3, 16 ug of recombinant chitinase; 4, 8 pg of recombinant chitinase. (B) The effects of the recombinant chitinase on the spore germination and hyphal growth of B. cinereu measured by the microspectrophotometric method. Relative growth = A,,, of sample microculture/A,,, of control microculture. Error bars represent SE (n=3).

mined by measuring the increase in reducing power of a reaction mixture (11, 12). A reaction mixture consisting of 1.0 ml of a substrate solution (0.1% glycolchitin in 50 mM sodium acetate buffer, pH 4.0) and 0.1 ml of the enzyme solution (5 pg/ml) was incubated at 37°C for 1 h. The reaction was stopped by adding 2 ml of 0.1% potassium ferricyanide in 0.5 M sodium carbonate. After boiling the reaction-terminated solution mentioned above for 15 min, the absorbance was measured at 420 nm. The effects of pH and temperature on the activity of the recombinant chitinase were measured. The optimum pH and temperature for recombinant chitinase activity were 4.0 and 40°C respectively (Fig. 2). The recombinant chitinase was stable between pH3.0-6.0 at 4°C and stable below 40°C at pH4.0 (Fig. 2). The inhibitory activity of the recombinant chitinase on the growth of Botrytis cinerea RIFY 4 115 was determined using a potato dextrose agar (PDA) plate (13) and by a microspectrophotometric method (14). A small block of PDA containing fungal hypha, which was excised from the edge

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of an actively growing culture, was placed at the center of the PDA plate and then incubated at 25°C. After the fungal colony diameter had reached 2.0 cm, sterile filter paper discs were laid on the agar surface and 100 ~1 of the sample solution was applied to the discs. The plate was further incubated at 25”C, and the growth of the fungal hypha was monitored visually (Fig. 3A). The microspectrophotometric method using a 96-well microplate was carried out as follows. The sample solution (40 ~1, O-175 pg.rnl-’ in PBS) and the fungal spore solution (160 ~1, 2 x lo3 spores.ml-’ in half-strength PDA broth) were pipetted into each well of a 96-well microplate and incubated for 24 h at 25°C in the dark, and then the absorbance of the wells was measured at 595 nm. Relative growth of B. cinerea was calculated using the following equation: relative growth = A,,, of sample microculture/A,,, of control microculture (Fig. 3B). Hyphal growth of B. cinerea on PDA plate was inhibited around the filter paper disc containing the recombinant chitinase and the extent of inhibition depended on the concentration of the recombinant chitinase (Fig. 3A). The relative growth of B. method cinerea measured by the microspectrophotometric decreased as the concentration of the recombinant chitinase increased (Fig. 3B). These results suggest that one potential role of this chitinase in the defense system of grapes is its direct attack on pathogenic fungi. This study was supported in part by a Grant-in-Aid for Scientific Researches (Bl4360018) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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