Molecular cloning,expression, and characterization of an endo-beta-1,4-glucanase cDNA from Epidinium caudatum1

An endo-beta-1,4-glucanase gene (epi3) from the rumen ciliated protozoan Epidinium caudatum was cloned from a cDNA library constructed by using the lambda ZAP II vector. The enzymatic activity of the gene product was detected by the Congo red assay, using carboxymethyl cellulose (CMC) as substrate. The nucleotide sequence of epi3 revealed 1,253 nucleotides with an open reading frame for a protein (Epi3) of 356 amino acids (Mr -41,014). Epi3 shows high homology with family 5 endoglucanase genes and with genes from protozoa isolated from sources other than the rumen. The specific activity of Epi3 produced in Escherichia coli was 5.544, 2.754, and 0.295 mmol of glucose min(-1) mg(-1) protein when the substrates used were CMC, beta-glucan, and xylan, respectively. A beta-1,4-linked trisaccharide of glucose was the preferred substrate of Epi3, as determined by analysis with the p-nitrophenyl form of the substrate. To our knowledge, this is the first report of the isolation of an endoglucanase gene from a rumen protozoan.     

Identification and fine mapping of AvrPi15, a novel avirulence gene of Magnaporthe grisea

Avirulence of Magnaporthe grisea isolate CHL346 on rice cultivar GA25 was studied with 242 ascospore progenies derived from the cross CHL346 × CHL42. Segregation analysis of the avirulence in the progeny population was in agreement with the existence of a single avirulence (Avr) gene, designated as AvrPi15. For mapping the Avr gene, we developed a total of 121 microsatellite DNA markers [simple sequence repeat (SSR)], which evenly distributed in the whole-genome of M. grisea through bioinformatics analysis (BIA) using the publicly available sequence. Linkage analysis of the AvrPi15 gene with these SSR markers showed that six markers on chromosome 6, MS6-1, MS6-2, MS6-3, MS6-7, MS6-8 and MS6-10, were linked to the AvrPi15 locus. To further define the chromosomal location of the AvrPi15 locus, two additional markers, MS6-17 and STS6-6, which were developed based on the sequences of telomeric region 11 (TEL11), were subjected to linkage analysis. The results showed that MS6-17 and STS6-6 were associated with the locus by 3.3 and 0.8 cM, respectively. To finely map the Avr gene, two additional candidate avirulence gene (CAG) markers, CAG6-1 and CAG6-2, were developed based on the gene annotation of the sequence of TEL 11. Linkage analysis of the Avr gene with these two markers revealed that both of them completely cosegregated with the AvrPi15 locus. Finally, this locus was physically mapped into ∼ 7.2-kb interval of the TEL11 by BIA using these sequence-ready markers. This is the key step toward positional cloning of the AvrPi15 gene.     

稻瘟病拮抗菌株的分离、筛选及鉴定

从水稻病健叶、茎和根组织以及稻田土壤中共分离得到细菌菌株321株。经发酵法初筛,对稻瘟病菌丝生长有抑制作用的有57株,再通过平板对峙法复筛,具有强烈拮抗作用的菌株有5株,其抑菌距离达16mm以上。分别对5个菌株进行形态学观察、生理生化指标进行鉴定,结果有1株(No.156)为Bacillus subtilis,2株(No.171和No.177)为Bacillus pumillus,2株(No.192和No.279)为Bacillus ploymyxa。     

稻曲病菌PMK1类同源基因克隆及在稻瘟病菌遗传互补中的功能验证

[目的]克隆稻曲病菌PMK1类MAPK(Mitogen-activated protein kinase)同源基因.[方法]根据丝状真菌MAPK蛋白保守性设计简并引物扩增稻曲病菌MAPK基因部分片段,进而利用TAIL-PCR进行染色体步移和RT-PCR获得UVMK1基因全长和cDNA全长.构建互补载体,交叉互补稻瘟病菌APMK1突变体菌株nn78进行功能验证,包括附着胞分化和致病性测定.[结果]UVMK1基因全长1435 bp,包含3个内含子,编码355氨基酸的蛋白.UVMK1推导蛋白与丝状真菌Magnaporthe grisea PMK1,Fusarium oxysporum FMK1,Fusarium solani FSMAPK,Colletotrichumlagenarium CMK1,Botrytis cinerea BMK1,Claviceps purpurea CMPK1等编码蛋白高度同源.转化稻瘟病菌菌株nn78,获得5个转化子.其中选取的转化子恢复了稻瘟病菌正常的附着胞分化和对大麦叶片的致病能力.[结论]本研究成功分离了首个稻曲病菌MAPK基因,而且UVMK1基因是稻瘟病菌PMK1的同源基因.     

Gene discovery and gene expression in the rice blast fungus, Magnaporthe grisea: analysis of expressed sequence tags

Over 28,000 expressed sequence tags (ESTs) were produced from cDNA libraries representing a variety of growth conditions and cell types. Several Magnaporthe grisea strains were used to produce the libraries, including a nonpathogenic strain bearing a mutation in the PMK1 mitogen-activated protein kinase. Approximately 23,000 of the ESTs could be clustered into 3,050 contigs, leaving 5,127 singleton sequences. The estimate of 8,177 unique sequences indicates that over half of the genes of the fungus are represented in the ESTs. Analysis of EST frequency reveals growth and cell type-specific patterns of gene expression. This analysis establishes criteria for identification of fungal genes involved in pathogenesis. A large fraction of the genes represented by ESTs have no known function or described homologs. Manual annotation of the most abundant cDNAs with no known homologs allowed us to identify a family of metallothionein proteins present in M. grisea, Neurospora crassa, and Fusarium graminearum. In addition, multiply represented ESTs permitted the identification of alternatively spliced mRNA species. Alternative splicing was rare, and in most cases, the alternate mRNA forms were unspliced, although alternative 5' splice sites were also observed.     

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4- glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion- exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.      

Cloning, high-level expression, purification, and properties of a novel endo-beta-1,4-mannanase from Bacillus subtilis G1 in Pichia pastoris

A novel gene coding for an endo-beta-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The beta-mannanase showed an identity of 90.2-92.9% (< or =95%) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified beta- mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDITOF mass spectrometry. The recombinant beta-mannanase had an optimum temperature of 45 degrees C and optimum pH of 6.5. The enzyme was stable at temperatures up to 50 degrees C (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions (Hg2+, Pb2+, and Co2+) substantially inhibited the recombinant beta-mannanase. The chemical additives including detergents (Triton X- 100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the beta-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.     

稻瘟菌糖蛋白激发子(CSBI)的纯化及其鉴定

稻瘟菌（Magnaporthe grisea）ZC1l3菌株97-151a菌丝经离心、超滤、Sephacryl S-100凝胶柱、DEAE-Sepharose FF阴离子交换柱层析,纯化获得糖蛋白激发子CSBI。CSBI经SDS-PAGE后银染显示单一条带,糖,蛋白比例约为9．32。CSBI对非亲和性互作水稻叶片中过氧化物酶的诱导显著高于亲和性互作水稻（P〈0．05）。经N端氨基酸同源序列比对表明,CSBI与MG07877．4推测蛋白的同源性最高。经基质辅助激光解析电离飞行时间质谱鉴定也表明CSBI是该推测蛋白。     

Identification of a putative vacuolar serine protease gene in the rice blast fungus,Magnaporthe grisea

We identified and cloned a gene designated SPM1, encoding a serine protease from the rice blast fungus Magnaporthe grisea. SPM1 is a single-copy gene, encoding a subtilisin-like serine protease with 536 amino acids. Analyses of the deduced amino acid sequence of SPM1 suggested that SPM1 would be localized in a vacuole, an important organelle in pathogenicity.     

稻瘟病菌致病突变体的REMI诱变与鉴定

以水稻“爱知旭”(Aichi—aShahi)为寄主,将带选择标记的质粒pCSN43和pBF101为外源DNA,利用限制酶诱导整合(REMI)这种新方法转化稻瘟病菌原生质体,从筛选到的数百个转化体中分离出3个与致病能力密切相关的突变体R2H65、R2H69和R281565。其中,R2H65和R2H69只产生畸形分生孢子,分生孢子的发育和附着胞的形成以及黑色素的合成均受到极大影响,致病性测试证明完全丧失致病能力;R281565的许多表型与野生型的相似,但致病能力却大大降低。     

Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea

Proteomic approaches using two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from rice leaf that are differentially expressed in response to the rice blast fungus, Magnaporthe grisea. Microscopic observation of inoculated leaf with M. grisea revealed that callose deposition and hypersensitive response was clearly visible in incompatible interactions but excessive invading hypha with branches were evident in compatible interactions. Proteins were extracted from leaves 24, 48, and 72 hours after rice blast fungus inoculation. Eight proteins resolved on the 2-DE gels were induced or increased in the inoculated leaf. Matrix-assisted laser desorption/ionization-time of flight analysis of these differentially displayed proteins showed them to be two receptor-like protein kinases (RLK), two beta-1.3-glucanases (Glu1, Glu2), thaumatin-like protein (TLP), peroxidase (POX 22.3), probenazole-inducible protein (PBZ1), and rice pathogenesis-related 10 (OsPR-10). Of these proteins, RLK, TLP, PBZ, and OsPR-10 proteins were induced more in the incompatible interactions than in compatible ones. A phytohormone, jasmonic acid also induced all eight proteins in leaves. To confirm whether the expression profile is equal to the 2-DE data, seven cDNA clones were used as probes in Northern hybridization experiments using total RNA from leaf tissues inoculated with incompatible and compatible rice blast fungal races. The genes encoding POX22.3, Glu1, Glu2, TLP, OsRLK, PBZ1, and OsPR-10 were activated in inoculated leaves, with TLP, OsRLK, PBZ1, and OsPR-10 being expressed earlier and more in incompatible than in compatible interactions. These results suggest that early and high induction of these genes may provide host plants with leading edges to defend themselves. The localization of two rice PR-10 proteins, PBZ1 and OsPR-10, was further examined by immunohistochemical analysis. PBZ1 accumulated highly in mesophyll cells under the attachment site of the appressorium. In contrast, OsPR-10 expression was mainly localized to vascular tissue.     

Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

Isolation, purification and characterization of an antifungal molecule produced by Bacillus licheniformis BC98, and its effect on phytopathogen Magnaporthe grisea

AIMS: Isolation of bacterial antagonist for use in the biological control of phytopathogenic fungi like rice blast fungus, Magnaporthe grisea, and to further purify and characterize the antifungal molecule produced by the antagonist. METHODS AND RESULTS: Bacterial antagonist exhibiting highest antifungal activity against the rice blast fungus M. grisea was isolated from soil and identified as Bacillus licheniformis BC98. Besides M. grisea, the isolate also inhibited the growth of other phytopathogens such as Curvularia lunata and Rhizoctonia bataticola. Biologically active fractions were isolated from the culture filtrate and further fractionated by reverse-phase high-performance liquid chromatography (HPLC) enabling detailed structural characterization of a component of molecular mass 1035 Da. The active peptide was identified as surfactin after 500 MHz (1)H NMR analysis. Microscopic analysis of the effect of the antagonist on M. grisea revealed bulbous hyphae showing patchy and vacuolated cytoplasm when observed under the electron microscope. CONCLUSIONS: The antagonistic lipopeptide secreted by B. licheniformis BC98 and identified as surfactin, induced morphological changes in M. grisea, inhibiting its further growth, and thus exhibiting fungicidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonist inhibits germination of M. grisea, a potent rice phytopathogen, and therefore appears to be a potential candidate for control of rice blast disease.     

Inexpensive, rapid procedure for bulk purification of cellulase-free beta-1,4-D-xylanase of high specific activity

A process has been developed for the bulk purification of cellulase-free beta-1,4-D-xylanase from the fungus Trichoderma harzianum E58. The process involved the primary step of ultrafiltering the culture filtrate via a 10,000-molecular-weight cut-off membrane to separate the cellulase (retentate) and xylanase (permeate) fractions. The cellulase component was concentrated by 40- to 60-fold, resulting in an enzyme complex that could effectively hydrolyze high concentrations of cellulose and xylan to glucose and xylose. The xylanase was concentrated and solvent exchanged by adsorption to a cationic exchanger, SP-ZetaPrep 250, followed by elution with a pH change in the buffer to give a purified and concentrated xylanase complex dissolved in a low-salt buffer. The resultant xylanase system was pure by the criteria of sodium dodecyl sulfate polyacrylamide electrophoresis, had a very high specific activity of 2400 IU/mg protein, was virtually free of filter paper activity, and had a ratio of contaminating filter paper activity of 2 x 10(-6) (0.009% endoglucanase activity). Approximately 3.3 g protein, which contained in excess of 7 x 10(6) IU xylanase activity, was obtained from 17 L original culture filtrate. The process scheme was designed to facilitate scale-up to an industrial level of production.