枯草芽孢杆菌XF-1中脱乙酰几丁质酶在大肠杆菌中的表达及其抑茵活性

根据脱乙酰几丁质酶同源基因序列设计引物,扩增、克隆和测序了枯草芽孢杆菌Bacillussubtilis菌株XF-1的脱乙酰几丁质酶基因（CSrlo该基因编码区为834bp,编码由277个氨基酸组成的约30kD的蛋白质,其基因序列与枯草芽孢杆菌菌株B168的脱乙酰几丁质酶基因的相似性达到99％,氨基酸序列相似性为98％,在第19、47、60、256和257位的氨基酸发生了变化,分别由异亮氨酸代替了菌株B168脱乙酰几丁质酶中的甲硫氨酸、缬氨酸代替了谷氨酸、苏氨酸代替了异亮氨酸、天冬氨酸代替了谷氨酸、赖氨酸代替了天冬酰胺。菌株xF一1对稻瘟病菌Magnaportheoryzae和芸薹根肿菌Plasmodiophorabrassicae有抑制作用,而菌株B168没有。菌株xF－1和B168的CSFI基因在大肠杆菌Escherichiacoli中的表达产物均具有脱乙酰几丁质酶活性,但前者的表达产物对稻瘟病菌及芸薹根肿菌具有抑制作用,而后者没有。表明脱乙酰几丁质酶是xF－1抑制根肿病作用机制之一,且上述5个氨基酸替代可能与抑菌机制有关。     

Rapid detection of chitosanase activity in chitosanase gene-transformed strains of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> by lytic infection of specific bacteriophages

 In combination with lytic infection by virulent phages, a simple method for monitoring transgenic strains of Enterobacter cloacae was developed in this study. First, 15 strains of E. cloacae were used as indicator bacteria to isolate virulent phages with different host ranges. Of the phages isolated, five isolates (EcP-22, -35, -45, -55, and -70) were used to construct a set of virulent phages corresponding to all strains of E. cloacae. Using this phage set, a rhizosphere strain (KRM-055E) of E. cloacae was effectively screened from field soil. KRM-055E was transformed with a prokaryotic chitosanase gene csnSM1 and infected with the phage EcP-03, which can lyse the strain most effectively. The lysis of KRM-055E/csn occurred 2 h after inoculation, and the chitosanase activity was simply detected by dropping the lysate onto an agar plate containing glycol chitosan. The positive signal for chitosanase activity was detected in the 2-h lysates, and the signal intensity reached a maximum in the 5-h lysate. The present assay was simple, rapid, inexpensive, easy to perform, and applicable to another strains. Received: August 2, 2002 / Accepted: October 31, 2002 Acknowledgments This work was supported in part by a grant (no. 99L01205) from the “Research for the Future” program of the Japan Society for the Promotion of Science. We are grateful to Dr. M. Sato, National Institute of Agrobiological Science, Dr. H. Okamoto, Fukui Agricultural Experiment Station, and Dr. K. Tsuda, Kyoto Prefectural Institute of Agricultural Biotechnology, for kindly providing E. cloacae strains. We thank Dr. P. Park, Kobe University, for technical support with the electron microscopic observations.     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.     

Purification, N-terminal amino acid sequencing and antifungal activity of chitinases from pepper stems treated with mercuric chloride

Different isoforms of chitinases were purified from pepper (Capsicum annuumL. cv. Hanbyul) stems treated with mercuric chloride. The acidic isoform a1 (69kDa, pI5.0), basic isoforms b1 (32kDa, pI9.0) and b2 (22kDa, pI9.1) were purified by chitin-affinity chromatography, with subsequent electroelution from nondenaturing polyacrylamide gel electrophoresis (PAGE) gels. The acidic isoform a1 has chitin-binding properties, but no antifungal activity. The basic isoforms b1 and b2 contain high ratios of cysteine and glycine at the N-terminal chitin-binding domain, exhibit chitinase activity, and show antifungal activities againstColletotrichum gloeosporioides, Fusarium oxysporumf.sp.cucumerinum, Magnaporthe grisea, andTrichoderma viride in vitro.Moreover, their antifungal activity shows a high degree of specificity to filamentous fungi. The chitinases b1 and b2 show a high sequence identity in their N-terminal residues with those from wheat, tobacco, potato, rice andArabidopsis thaliana.None of the purified isoforms of chitinases inhibited hyphal growth of the Oomycete fungus which lacks chitinPhytophthora capsici. In contrast, zoospore germination and germ tube elongation ofP. capsiciwere effectively inhibited by treatment with b1 and b2.     

Isolation of Olive latent virus 1 from Tulip in Toyama Prefecture

A virus whose coat protein gene had a high sequence homology with the coat protein gene of Olive latent virus 1 was isolated from diseased tulip in Toyama Prefecture. Received 1 June 2001/ Accepted in revised form 1 August 2001     

The Complete Nucleotide Sequence of a Japanese Isolate of White clover mosaic virus Strain RC

The complete nucleotide sequence was determined for genomic RNA of White clover mosaic virus (WClMV-RC) isolated from red clover (Trifolium pratense) in Japan, It is 5843 nucleotides in length, excluding the poly(A) tail at the 3' terminus. Similar to other potexviruses, it contains five open reading frames (ORFs 1 through 5), which putatively encode an RNA-dependent RNA polymerase (RdRp) (147 kDa), a triple gene block (TGB) (26 kDa/13 kDa/7 kDa), and a coat protein (CP) (22 kDa), respectively. The deduced amino acid sequence of the WClMV-RC CP was identical to that of WClMV-O, one of two New Zealand isolates, but only 85% identical to that of WClMV-M, the other New Zealand isolate, because of heterogeneity in the C-termini of CP amino acid sequences. The implication of this CP heterogeneity is discussed. Received 30 August 2001/ Accepted in revised form 11 January 2002     

Purification and partial characterization of xylanase from the fungal maize pathogenHelminthosporium turcicum (Pass)

The fungal pathogenHelminthosporium turcicum was found to secret xylanase when grown on minimal medium containing xylans, wheat straw or isolated maize cell walls. The highest xylanase activity occurre when the fungus was grown on maize cell walls. When glucose was added to this medium xylanase activity was suppressed. The xylanase enzyme was purified from the culture filtrate by subsequent anion exchange chromatography, cation exchange chromatography and gel filtration. The purified xylanase gave a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to an apparent molecular weight of 22.5 kDa. It is determined to have a pI of 7.4, specific activity of 11300 nanokatals mg^–1, pH optimum between pH 5.5 and 6.5 and optimal temperature between 50 °C and 60 °C. The half-life of the enzyme at pH 6.0 and 50 °C was found to be 35 min. For primary structure comparison with other xylanases, the protein was digested with trypsin and the resulting peptides were separated by reversed phase chromatography and selected peptides were sequenced. The determined amino acid sequence showed high homology with xylanase fromCochliobolus carbonum and three other fungal xylanases.     

Gene Arrangement and Sequence of str Operon of Phytoplasma Resemble Those of Bacillus More Than Those of Mycoplasma

The complete region of a putative streptomycin operon (str operon) of onion yellows (OY) phytoplasma, a phytopathogenic mollicute, was isolated and sequenced. This operon contains four genes, rps12, rps7, fus, and tuf, encoding ribosomal proteins S12 and s7, elongation factor (EF) -G, and EF-Tu, respectively. These four genes constitute the str operon in non-mollicute bacteria, such as Escherichia coli and Bacillus subtilis. In two species of mollicute Mycoplasma, the tuf gene was reported not to be included in this operon, but was located apart, indicating that the gene arrangement of this operon in phytoplasmas resembles that of B. subtilis more than that of Mycoplasma spp. In addition, the deduced amino acid sequence of EF-G of phytoplasmas also resembles that of B. subtilis more than that of Mycoplasma spp. These results suggest that analyses of the gene organization and sequence of the phytoplasma genome will provide valuable insights into evolutionary relationships among the culturable mollicutes, phytoplasmas and other Gram-positive bacteria. Received 25 April 2001/ Accepted in revised form 21 August 2001     

Purification and Partial Characterization of Figaren, an RNase-like Novel Antiviral Protein from Cucumis figarei

An antiviral protein, designated figaren, was purified from leaves of Cucumis figarei and partially characterized. Column chromatography, SDS-polyacrylamide gel electrophoresis and periodic acid-Schiff staining revealed that figaren is a glycoprotein with a molecular weight of 23 kDa. Figaren was stable at pH 2 to 12 and below 90°C. N-terminal amino acid sequencing indicated that figaren contained the conserved region for the S-allele-associated ribonucleases (RNases). In-gel RNase assay showed that figaren digested yeast RNAs. Figaren also digested double-stranded RNAs extracted from Cucumber mosaic virus (CMV)-infected tobacco tissues. Fluorescence in situ hybridization revealed that figaren and RNases (beef pancrease and RNase T₁ at 1 μg/ml similarly inhibited CMV infection in cowpea leaves. Figaren and the RNases at 5-500 ng/ml had similar inhibitory effect on local lesion formation by CMV. These data suggest that figaren is a novel RNase-like antiviral protein. Received 10 October 2000/ Accepted in revised form 6 February 2001     

Characterization of an Extracellular Serine Protease of Fusarium eumartii and its Action on Pathogenesis Related Proteins

Proteases have been proposed as part of the invasion strategies of some pathogenic fungi. In this work, a serine protease produced by the phytopathogenic fungus Fusarium solani f.sp. eumartii was purified and characterized. Purification of the enzyme was accomplished by gel filtration through a Superose 12 column, followed by hydrophobic interaction chromatography in Phenyl Superose and gel filtration chromatography through Superdex 75. Analysis of the purified enzyme by SDS/PAGE without heat treatment, revealed a single band, which corresponded to the proteolytic activity detected by zymogram. When this protein was subjected to denaturing conditions, two major polypeptides of approximately 30 and 33kDa were revealed. The N-terminal amino acid sequence of one of these polypeptides showed a high similarity with fungal mature serine proteases of the subtilisin family. This protease hydrolysed in vitro, specific polypeptides of potato intercellular washing fluids and cell walls. The protease was also able to degrade pathogenesis-related proteins from the intercellular washing fluids. The role of this serine protease as part of the fungal strategy to colonize potato tuber tissues is discussed.     

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (³⁶⁸Val to Phe, ³⁶⁹Gln to His, and ⁴⁴⁷Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (³⁶⁹Gln to Pro and ³⁷³Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.     

Purification of an Elicitor-Inducible Antifungal Chitinase from Suspension-Cultured Rice Cells

Suspension-cultured rice cells showed an appreciable amount of chitinase activity when the cultured cells were treated with an elicitor isolated fromRhizoctonia solani, the rice sheath blight pathogen. A fivefold increase in chitinase activity was observed 24 h after elicitor treatment. The elicitor-inducible chitinase was purified by ammonium sulfate fractionation, chitin affinity chromatography and gel filtration. Its molecular weight is 35 kDa and it has an isoelectric point of 8.3. The 35 kDa basic chitinase inhibited mycelial growth ofR. solani in vitro. Morphological changes appeared within 1 h following exposure of mycelium to the chitinase. The hyphal tip showed marked swelling and subsequently lysis was also observed.     

Identification and characterization of a tospovirus causing chlorotic ringspots on <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchids

A putative virus-induced disease showing chlorotic ringspots on leaves of Phalaenopsis orchids has been observed in Taiwan for several years. A virus culture, 91-orchid-1, isolated from a Phalaenopsis orchid bearing chlorotic ringspot symptoms was established in Chenopodium quinoa and Nicotiana benthamiana, and characterized serologically and biologically. The virus reacted slightly with the antiserum of Watermelon silver mottle virus (WSMoV) but not with those of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and Groundnut ringspot virus (GRSV). Isometric particles measuring about 70–100 nm were observed. Inoculation with isolated virus was conducted to confirm that 91-orchid-1 is the causal agent of chlorotic ringspot disease of Phalaenopsis orchids. To determine the taxonomic relationships of the virus, the conserved region of L RNA and the complete nucleocapsid gene (N gene) were cloned and sequenced. The sequence of conserved region of L RNA shares 83.8, 82.5, 64.4 and 64.9% nucleotide identities and 96.5, 97.7, 67.3 and 67.6% amino acid identities with those of Peanut bud necrosis virus (PBNV), WSMoV, TSWV and INSV, respectively, indicating that 91-orchid-1 is a tospovirus related to WSMoV. The complete nucleotide sequence of the N gene determined from a cDNA clone was found to be 828 nucleotides long encoding 275 amino acids. Sequence analyses of the N gene showed that 91-orchid-1 is an isolate of Capsicum chlorosis virus (CaCV) which has been reported to infect tomato and capsicum plants in Australia and Thailand. 91-orchid-1 is therefore designated as CaCV-Ph. To our knowledge, this is the first formal report of a tospovirus infecting Phalaenopsis orchids.     

Laccase Gene LAC1 of Colletotrichum lagenarium Is Not Essential for Melanin Biosynthesis and Pathogenicity

Laccase has been shown to oxidize 1,8-dihydroxynaphthalene (1,8-DHN) in the final step of melanin biosynthesis in several fungi. In this study, a laccase gene (LAC1) was cloned from Colletotrichum lagenarium that synthesizes 1,8-DHN melanin, and characterized. To clone the LAC1 sequences, genomic DNA was subject to polymerase chain reactions (PCR) with degenerate oligonucleotide primers that were designed on the basis of amino acid sequences conserved among characterized laccases from other ascomycetes Botrytis cinerea, Neurospora crassa, Aspergillus nidulans, and Cryphonectria parasiticus. The LAC1 gene contained an open reading frame composed of 589 codons and three introns of 51, 49, and 57 nucleotides. The deduced amino acid sequence of Lac1p had high similarity to that of laccase from N. crassa and significant homology with those of multicopper blue proteins. Under melanin-induced culture of this fungus, laccase activity significantly increased and LAC1 expression was also detected. However, the lac1Δ mutants retained laccase activity and had no significant phenotypic differences in melanin production or pathogenicity from the wild-type strain. Received 23 February 2001/ Accepted in revised form 29 March 2001     

Harpinpsta from Pseudomonas syringae pv. tabaci Is Defective and Deficient in Its Expression and HR-inducing Activity

To elucidate the role of harpins produced by Pseudomonas syringae, the corresponding hrpZ gene was isolated from P. s. pv. tabaci. The sequence information revealed that this gene carries a serious mutation with 326 bp lacking in the central region and potentially encodes only 140 N-terminal amino acids because of a frame shift. The investigation of biological properties using recombinant harpin indicated harpin_psta was incapable of inducing HR in both host and nonhost plants. Based on an immunoblot analysis to detect harpin from P. s. pathovars in hrp-inducing medium, the truncated harpin_psta was neither expressed nor secreted into the culture medium. These results suggest that harpin is not the sole determinant of the host-parasite specificity in P. s. pv. tabaci. Received 10 August 2000/ Accepted in revised form 21 December 2000     

Midgut juice of Plutellaxylostella highly resistant to Bacillusthuringiensis Cry1Ac contains a three times larger amount of glucosinolate sulfatase which binds to Cry1Ac compared to that of susceptible strain

Midgut juice of Plutellaxylostella strain PXR which is resistant to Cry1Ac was biochemically characterized relative to the susceptible PXS strain. The midgut juice of PXR (PXR-Juice) was shown to process Cry1Ac protoxin to 60 kDa active toxin with the same processing pattern as that of juice from PXS (PXS-Juice) in SDS–PAGE. PXS larvae which were given the Cry1Ac toxin pre-processed with PXR-Juice were killed with the same rate as that with Cry1Ac pre-activated by trypsin. PXR-Juice was found to contain three times larger amount of 66 kDa protein (P66) than PXS-Juice and the N-terminal amino acid sequence of P66 was matched to that of glucosinolate sulfatase in data base search. The protein band of P66 was coincided with the band of p-nitro phenyl sulfatase activity in zymogram. P66 purified to homogeneity in SDS-PAGE bound to Cry1Ac and soybean agglutinin, and K_D for Cry1Ac was estimated to be 718 nM with surface plasmon resonance analysis. Using purified sulfatase, K_m and V_max were estimated and involvement of the enzyme in the PXR resistance was discussed.     

Fusarium moniliforme secretes four endopolygalacturonases derived from a single gene product

Extracellular endopolygalacturonase, purified from the pathogenic fungus Fusarium moniliforme, consists of four molecular forms (38, 41·5, 45, and 48·5 kDa, respectively. Three forms (38, 41·5, and 45 kDa) were purified to homogencity by FPLC on a Mono S column followed by electroelution after SDS-PAGE. The N-terminal amino acid sequences of each of the three forms, and of a mixture containing all four forms were shown to be identical to that predicted from the nucleotide sequence of the endopolygalacturonase gene previously cloned from F. moniliforme. Enzymatic deglycosylation experiments revealed the presence of N-linked, high mannose oligosaccharide side-chains on all four forms of endo polygalacturonase. Hydrogen fluoride catalysed chemical deglycosylation of the polygalacturonase mixture yielded a single polypeptide with an apparent molecular mass of 36·2 kDa. Southern blot analysis, carried out at high stringency with an endopolygalacturonase-specific probe on genomic DNA digested with three different restriction enzymes, showed a single hybridizing restriction fragment in all three digests. A single 2·0 Mb chromosome hybridized with the endo polygalacturonase-specific probe, as shown by Southern blot analysis of F. moniliforme chromosomes separated by CHEF electrophoresis. Northern blot analysis revealed only one mRNA species 1350 nt encoding endo polygalacturonase. These data indicate that a single gene encodes the endopolygalacturonases of F. moniliforme.     

Identification and characterization of a potyvirus causing chlorotic spots on <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchids

A putative virus-induced disease showing chlorotic spots on leaves of Phalaenopsis orchids was observed in central Taiwan. A virus culture, phalaenopsis isolate 7-2, was isolated from a diseased Phalaenopsis orchid and established in Chenopodium quinoa and Nicotiana benthamiana. The virus reacted with the monoclonal antibody (POTY) against the potyvirus group. Potyvirus-like long flexuous filament particles around 12–15 × 750–800 nm were observed in the crude sap and purified virus preparations, and pinwheel inclusion bodies were observed in the infected cells. The conserved region of the viral RNA was amplified using the degenerate primers for the potyviruses and sequence analysis of the virus isolate 7-2 showed 56.6–63.1% nucleotide and 44.8–65.1% amino acid identities with those of Bean yellow mosaic virus (BYMV), Beet mosaic virus (BtMV), Turnip mosaic virus (TuMV) and Bean common mosaic virus (BCMV). The coat protein (CP) gene of isolate 7-2 was amplified, sequenced and found to have 280 amino acids. A homology search in GenBank indicated that the virus is a potyvirus but no highly homologous sequence was found. The virus was designated as Phalaenopsis chlorotic spot virus (PhCSV) in early 2006. Subsequently, a potyvirus, named Basella rugose mosaic virus isolated from malabar spinach was reported in December 2006. It was found to share 96.8% amino acid identity with the CP of PhCSV. Back-inoculation with the isolated virus was conducted to confirm that PhCSV is the causal agent of chlorotic spot disease of Phalaenopsis orchids in Taiwan. This is the first report of a potyvirus causing a disease on Phalaenopsis orchids.