Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Dactylella shizishanna

AIMS: To evaluate the production of an extracellular serine protease by Dactylella shizishanna and its potential as a pathogenesis factor. METHODS AND RESULTS: An extracellular alkaline serine protease (Ds1) was purified and characterized from the nematode-trapping fungus D. shizishanna using cation-exchange chromatography and hydrophobic interaction chromatography. The molecular mass of the protease was approximately 35 kDa estimated by SDS-PAGE. The optimum activity of Ds1 was at pH 10 and 55 degrees C (over 30 min). The purified protease could degrade purified cuticle of Penagrellus redivivus and a broad range of protein substrates. The purified protease was highly sensitive to phenylmethyl sulfonyl fluoride (PMSF) (0.1 mmol l(-1)), indicating it belonged to the serine protease family. The N-terminal amino acid residues of Ds1 are AEQTDSTWGL and showed a high homology with Aozl and PII, two serine proteases purified from the nematode-trapping fungus Arthrobotrys oligospora. CONCLUSIONS: Nematicidal activity of D. shizishanna was partly related to its ability to produce extracellular serine protease. SIGNIFICANCE AND IMPACT OF THE STUDY: In this report, we purified a new serine protease from D. shizishanna and provided a good foundation for future research on infection mechanism.     

Cloning and characterization of an extracellular serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Arthrobotrys conoides</Emphasis>

An extracellular serine protease (Ac1) with a molecular mass of 35 kDa was purified from the nematode-trapping fungus Arthrobotrys conoides. The optimum activity of Ac1 is at pH 7.0 and 53.2°C (over 20 min). Ac1 can degrade a broad range of substrates including casein, gelatin, bovine serum albumin, collagen, and nematode cuticles. Moreover, the enzyme can immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, indicating Ac1 may be involved in infection against nematodes. The encoding gene of Ac1 contains one intron of 60-bp and two exons encoding a polypeptide of 411 amino acid residues. The deduced polypeptide sequence of Ac1 showed a high degree of similarity to two previously reported serine proteases PII and Mlx from other nematode-trapping fungi (81% aa sequence identity). However, three proteases Ac1, Aoz1 and Mlx showed optimum temperatures at 53.2, 45 and 65°C, respectively. Compared to PII, Ac1 appears to have a significantly higher activity against gelatin, bovine serum albumin, and non-denatured collagen. Moreover, our bioassay experiments showed that Ac1 is more effective at immobilizing P. redivivus than B. xylophilus.     

Purification and cloning of a novel serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Dactylellina varietas</Emphasis> and its potential roles in infection against nematodes

From the culture filtrate of the fungus Dactylellina varietas (syn. Dactylella varietas), an extracellular protease (designed Dv1) was purified by cation exchange and hydrophobic interaction chromatography. The purified protease showed a molecular mass of approximately 30 kDa and displayed an optimal activity at pH 8 and 60.5°C (more than 20 min). This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. However, its proteolytic activity was highly sensitive to the serine protease inhibitor Phenylmethylphonylfuoride (1 mM), indicating that it belongs to the serine-type peptidase group. This protease could immobilize the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans and hydrolyze the purified cuticle of P. redivivus, suggesting it may play a role in infection against nematodes. The encoding gene of Dv1 and its promoter sequence were cloned using degenerate primers and the DNA walking technology. Its open-reading frame contains 1,224 base pairs and without any intron. The deduced amino-acid sequence shared low identity to serine proteases from other nematode-trapping fungi. Our report identified a novel pathogenic protease from the nematode-trapping fungus D. varietas, and the three-dimensional structure of this protease was predicted using the Swiss-Prot method. Jinkui Yang and Lianming Liang contributed equally to this work.     

Purification and cloning of an extracellular serine protease from the nematode-trapping fungus Monacrosporium cystosporium

An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and 56 degrees (over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant (Km) and Vmax for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were 1.67x10-4 M and 0.6071 OD410 per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might play a role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mc1 showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mc1 possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.     

Characterization of a neutral serine protease and its full-length cDNA from the nematode-trapping fungus Arthrobotrys oligospora

A neutral serine protease (designated Aoz1) was purified to homogeneity from a strain of Arthrobotrys oligospora, obtained from soil in Yunnan Province. The purified protein showed a molecular mass of approximately 38?000 Dalton, pI 4.9 and displayed optimal activity at 45 C and pH 6-8. The protein could hydrolyze gelatin, casein and the chromogenic substrate azocoll, and it could immobilize nematodes in vitro (Panagrellus redivivus L. [Goodey]). The level of activity in culture medium was found to increase with increasing gelatin concentration. Scanning electron micrographs demonstrated dramatic structural changes in nematode cuticle treated with the purified protease. A partial peptide sequence obtained by N-terminal sequence analysis was used to design degenerate primers for the isolation of a cDNA gene encoding the mature protease. Analysis of the cDNA and corresponding genomic sequence revealed 97% identity with PII, a gene previously described from A. oligospora, and we conclude that this gene is likely a PII ortholog.     

Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation

Nematode-trapping fungi are "carnivorous" and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.     

Laboratory experiments to evaluate the ability of Arthrobotrys oligospora to destroy infective larvae of Cooperia species, and to investigate the effect of physical factors on the growth of the fungus

Laboratory investigations were designed to study the influence of temperature, pH and oxygen tension on the growth of Arthrobotrys oligospora, a nematode-trapping microfungus. Experiments were performed to evaluate the potential role of A. oligospora in destroying third-stage larvae of Cooperia spp. on agar plates and in cattle faeces. The fungus had a growth rate optimum at 23 degrees C and pH 6. Anaerobic cultivation for 23 hours at 23 degrees C and 39 degrees C inhibited fungal growth, but it did not destroy the fungus, which regained growth upon a subsequent shift to aerobic conditions at 23 degrees C. Under experimental conditions in petri-dishes containing agar, the nematode-trapping efficiency of the fungus was striking in that 100% of a population of third-stage larvae of Cooperia spp. was captured within three days of the experiment. The trapping efficiency in faeces was shown to depend upon the inoculation level. At a concentration of approximately 2500 conidia per g faeces, 99% of the larvae were destroyed. The possibilities of using nematode-trapping fungi in controlling animal-parasitic nematodes are discussed.     

Thioredoxin1 regulates conidia formation,hyphal growth,and trap formation in the nematode-trapping fungus Arthrobotrys oligospora

Arthrobotrys oligospora, a model nematophagous fungus that produces specific adhesive networks to capture nematodes, has been proposed as a potentially effective biological agent to control harmful plant-parasitic nematodes. Although thioredoxin has been characterized as playing important roles in many cellular processes in other species, its function in nematophagous fungi has not been studied. Here, the function of a thioredoxin homolog, Aotrx1, was investigated in A. oligospora. The encoding gene of Aotrx1 in the nematophagous fungus A. oligospora was knocked out by homologous recombination; strain growth was assessed. The ΔAotrx1 strain of A. oligospora showed a significant decrease in growth rate on different media (PDA, CMY, and TG), a 70% decrease of conidia production, and a lower germination rate compared with the wild type. The mutant strain was unable to form traps to capture nematodes and was more sensitive to SDS and H2O2. Thioredoxin is involved in conidia development, trap formation, normal mycelial growth, and resistance to environmental stresses in the nematode-trapping fungus A. oligospora.     

Deletion of a lectin gene does not affect the phenotype of the nematode-trapping fungus Arthrobotrys oligospora

A number of filamentous fungi are known to produce high levels of saline-soluble and low-molecular-mass lectins. The function of these proteins are not clear but it has been proposed that they are involved in storage of nutrients, development, recognition of other organisms, and defense reactions. A gene encoding such a lectin (AOL) was deleted in the nematode-trapping fungus Arthrobotrys oligospora by homologous recombination. The deletion mutants did not express any hemagglutinating activity or protein cross-reacting with AOL antibodies. There were no significant differences between the DeltaAOL and wild-type strains in spore (conidia) germination, saprophytic growth, and pathogenicity. Furthermore, there was no significant difference in the growth and reproduction of collembolan feeding on the various strains of A. oligospora. Thus either the previous proposed functions of AOL are not correct, or the fungus can compensate for the absence of the lectin by expressing other proteins with similar function(s) as AOL.     

Purification and characterization of a surface lectin from the nematode-trapping fungus Arthrobotrys oligospora.

Several studies have indicated that the capture of nematodes by the nematophagous fungus Arthrobotrys oligospora is mediated by a lectin on the fungal surface. One of the major surface proteins of this fungus showed haemagglutinating activity and was isolated by affinity chromatography using a mucin Sepharose column. Biochemical analysis showed that the protein was a dimeric glycoprotein with a molecular mass of 36 kDa and an isoelectric point of pH 6.5, and contained no sulphur amino acids. The protein was N-terminally blocked; four internal peptides were sequenced, and showed no significant similarity to sequences in the Swiss-Prot or PIR databases. The haemagglutinating activity of the isolated protein was not inhibited by any of the mono- or disaccharides tested, but it was inhibited by the glycoproteins fetuin and mucin. The haemagglutinating activity changed after incubating the protein in buffers of different pH, with maximal activity at pH 11.0 and no activity at pH 2.8. The lectin was tested for different enzymic activities but none were detected. Analysis of the haemagglutinating activity in various cell fractions indicated that the protein was associated with extracellular polymer layers and with the cell wall of the fungus. About the same amount of the haemagglutinating protein was recovered from samples of vegetative mycelium and of mycelium containing nematode-trapping cells.     

A fungal lectin and its apparent receptors on a nematode surface

Abstract A lectin was isolated from the nematode-trapping fungus Arthrobotrys oligospora . This carbohydrate-binding protein was developmentally regulated and was found only on trap-bearing mycelia. The lectin receptor on the surface of the nematode Panagrellus redivivus has, furthermore, been investigated using homogenates from whole nematodes or nematode cuticle. The ability of macromolecules from nematodes, fractionated according to M _r and lectin affinities, to inhibit the capture of nematodes by A. oligospora was tested using an inhibition assay, based on a simple dialysis membrane technique. One major lectin-binding glycoprotein, with apparent M _r of 65 000, was isolated from the nematode cuticle.     

捕食线虫真菌在北京地区果园中的季节性分布

通过对北京海淀区2个苹果园和2个葡萄园根际捕食线虫真菌2年的季节性变化调查,共分离到199个捕食线虫真菌菌株,隶属3个属16个种,Arthrobotrysdactyloides分离频率最高,其次是A.oligospora,A.conoides和Stylopagesp.。捕食线虫真菌的数量在晚春夏初最丰富,秋季次之,冬夏最少。通过统计分析,捕食线虫真菌数量的季节性变化与温度线性相关,而与线虫总数S形相关,与土壤湿度和植物寄生线虫数量无显著相关。     

Morphogenesis and significance of hyphal coiling by nematode-trapping fungi in mycoparasitic relationships

Abstract The mycoparasitic behaviour of some nematode-trapping fungi was investigated. These organisms interacted with other soil fungi by hyphal coiling around the host hyphae. A detailed study with Arthrobotrys oligospora revealed that different fungi, representing all taxonomic groups, were attacked.
In dual cultures, the interaction between A. oligospora and Rhizoctonia solani occurred soon after hyphal contact, irrespective of the nutrient level of the medium. Coiling was also observed when the two organisms were grown in sterilized soil. The coils possessed a high metabolic activity compared to the surrounding hyphae, as was indicated by fluorescence microscopy of fluorescein diacetate (FDA)-stained preparations. On the ultrastructural level, developing coils showed an abundance of membranous vesicles which developed from tubular-shaped endoplasmic reticulum. At the site of coiling, a strong cell wall proliferation was observed in the Rhizoctonia cells. The cytoplasm of these cells subsequently disintegrated. The death of the cells was confirmed in vital staining experiments. Penetration of intact Rhizoctonia cells was not observed. The interaction between A. oligospora and R. solani is interpreted in terms of competition for nutrients.     

捕食线虫丝孢菌的菌寄生性测定

研究了节丛孢Arthrobotrys、单顶孢Monacrosporium和隔指孢Dactylella三个捕食线虫丝孢菌属16个菌株,对水稻立枯丝核菌RhizoctoniasolaniAG1、大豆核盘菌Sclerotiniasclerotiorum、茄科镰刀菌Fusariumsolani和恶疫霉Phytophthoracactorum四种常见土壤植物病原真菌的菌寄生性。结果表明供试菌可以通过弹簧式菌丝圈缠绕、类附着胞结构吸附、简单的菌丝缠绕或者贴附寄主菌丝生长四种方式寄生病原菌。其中,绝大多数菌株对立枯丝核病菌有寄生作用,一些供试真菌对其它三种病原真菌有寄生现象。利用孢子液浸泡法测定了其中5种捕食线虫真菌对核盘菌菌核的寄生能力,显示有较高寄生率。     

捕食线虫真菌少孢节丛孢Arthrobotrys oligospora线粒体基因组的异质性

捕食线虫真菌少孢节丛孢Arthrobotrys oligospora是子囊菌中的一个食肉性丝状真菌,其菌丝体可变态为各种结构精巧的捕食器官用于捕食线虫,在生物防治和进化上极具研究潜力.线粒体基因细胞色素c氧化酶亚基Ⅰ基因(COX1)在真菌物种鉴定中的效果饱受争议.本研究对A.oligospora的不同单孢培养的菌株进行...     

Induction of Nematode-Trapping Organs in the Predacious Fungus Arthrobotrys oligospora (Hyphomycetales) by Infective Larvae of Ostertagia ostertagi (Trichostrongylidae)

Laboratory experiments were designed to study the influence of temperature, concentrations of nematodes, oxygen tension, light, and nutrient levels, on the induction of nematode-trapping hyphal nets in the predacious fungus Arthrobotrys oligospora. When induced by infective Ostertagia ostertagi larvae, a maximum number of nets was produced at 20°C, at which temperature nets in surplus were produced at larval concentrations up to 1,000 larvae per cm2. A. oligospora did not produce nets in an anaerobic atmosphere containing 21 % CO2 (v/v), and net induction was suppressed to a certain degree by exposure to light. The composition of the medium had an important influence on the saprophytic growth and the net-forming capability of A. oligospora as a maximum number of nets was induced at a relatively low concentration of corn meal supporting the relatively sparse mycelium. It was shown that a proportion of trapping nets in A. oligospora maintained their trapping potential for more than 7 weeks when the temperature was below 25°C. Induction of nematodetrapping organs in A. oligospora is discussed in relation to control of infective nematode parasite larvae in cow pats.     

Fungal parasitism of the cyst nematode Heterodera schachtii: infection and enzymatic activity

Abstract Fungal egg parasites isolated from eggs of the cyst nematode Heterodera avenae in Sweden were investigated with respect to their ability to infect cyst nematode eggs of H. schachtii in vitro. The infection was studied by interference phase contrast microscopy of whole cysts and of cryosections of cysts exposed to the fungi on agar plates.
Verticillium suchlasporium was the most effective parasite, infecting 53% of the nematode eggs, while V. chlamydosporium infected 12% of the eggs. The fungi Paecilomyces lilacinus, Cylindrocarpon destructans or Fusarium oxysporum did not parasitize nematode eggs; nor did Arthrobotrys oligospora , a nematode trapping fungus nor Penicillium viridicatum which served as a control fungus.
The ability of the fungi to infect eggs was correlated with their lytic enzyme activity. Fungi that readily infected eggs also showed chitinase activity and presence of proteolytic activity. The Verticillium species had an activity between 3.7 and 14.6 μmol N -acetyl-glucosamine per mg protein per hour (CU) while it was 4.5 CU or lower for P. lilacinus . Other isolates did not shown any chitinase activity.     

食线虫真菌基因组测序和分析研究进展

食线虫真菌作为线虫的天敌,是一种潜在的线虫病害的生防制剂。食线虫真菌通过形成特殊的捕食器官或产生粘性孢子和毒素等方式捕捉和侵染线虫。近年来,随着测序技术的进步和生物信息学的应用,越来越多真菌基因组已经被测定和报道。目前,已经有7种食线虫真菌的基因组被报道,包括捕食线虫真菌寡孢节丛孢(Arthrobotrys oligospora)、线虫卵寄生真菌厚垣普可尼亚菌(Pochonia chlamydosporia)和内寄生真菌明尼苏达被毛孢(Hirsutella minnesotensis)等。本文对食线虫真菌的基因组特点、毒力相关基因家族的扩张、捕食器官形成调控和进化机制进行了系统地总结,对组学时代食线虫真菌研究面临的关键问题进行了评述。