Cloning, Sequencing and Expression of a Xylanase Gene from the Maize Pathogen Helminthosporium Turcicum

A gene encoding an endoxylanase from the phytopathogenic fungus Helminthosporium turcicum Pass. was cloned and sequenced. The entire nucleotide sequence of a 1991 bp genomic fragment containing an endoxylanase gene was determined. The xylanase gene of 795 bp, interrupted by two introns of 52 and 62 bp, encoded a protein of 227 amino acids showing up to 95% amino acid homology with other fungal xylanases. The precise splicing site of the introns was identified by sequencing the corresponding cDNA. A northern blot showed that the gene is expressed when the fungus is grown in a medium containing xylan as a sole carbon source. The cloned xylanase gene was expressed in maize plants during infection.     

The Fusarium graminearum Xyr1 transcription factor regulates xylanase expression but is not essential for fungal virulence

The fungal pathogen Fusarium graminearum is the causal agent of fusarium head blight in wheat and other small grain cereals. This fungus is known to produce high amounts of cell wall‐degrading enzymes during infection of wheat spikes. In addition, wheat tissue is particularly rich in xylan, which can be hydrolysed by fungal xylanases. In order to establish the role of F. graminearum xylanase activity in pathogenicity, targeted gene disruption of the F. graminearum xyr1 gene, encoding the major regulator of xylanase gene expression, was performed. When grown on xylan as carbon source, the xylanase activity of the Δxyr1 mutant was dramatically reduced and fungal growth was significantly reduced compared to the wildtype fungus. When grown on carboxymethylcellulose, the cellulolytic activity of the mutant was also reduced and the mutant did not grow on wheat cell walls. The disruption of the xyr1 gene greatly reduced the expression of xylanase‐encoding genes both in vitro and during wheat spike infection, thus confirming the involvement of F. graminearum Xyr1 in the regulation of genes controlling xylan degradation. However, despite the deep impact caused by xyr1 gene disruption on the expression of xylanase genes and on total xylanase activity, the virulence of the Δxyr1 mutant appeared unaffected on Triticum aestivum and T. durum spikes and on soybean seedlings. In conclusion, although a possible role for residual xylanase activity in the virulence of F. graminearum cannot be conclusively excluded, the results question the importance of xylanase activity during the infection process.     

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.     

Identification of hydrolytic activities expressed by Aspergillus flavus grown on cotton carpel tissue

To identify proteins important for invasion into host plant tissue, Aspergillus flavus was cultured on medium containing cotton carpel tissue as the sole carbon source. We identified several hydrolases suggesting they are important as A. flavus virulence factors for plant colonization. Specifically, Aspergillus flavus AF13 secreted at least two endoxylanase activities and a pectolytic activity when grown on the cotton carpel tissue medium. A concentrated sample derived from the A. flavus growth medium (6-day) was subjected to gel filtration chromatography on a BioGel P-30 column. A major endoxylanase activity was separated from the other fungal-secreted proteins. Additional fungal secreted proteins were partially resolved by gel filtration chromatography on a BioGel P-60 column. Multiple proteins with molecular weights in the 20 to 70 kD range were present in the harvested fungal growth medium. Analysis of these fungal-secreted proteins by liquid chromatography/tandem mass spectrometry identified both endo- and exo-glucanase proteins, α-l-arabinofuranosidase, glucoamylase, α-amylase A, pectate lyase A, xylanase F1, acetylxylan esterase, glutaminase A, as well as conserved hypothetical proteins of unknown function. These proteins likely assist A. flavus in the maceration of plant cell walls, allowing for pathogenic entry and accession of host nutrient resources. Pectolytic and xylanolytic hydrolases, as well as glucanases, appear to be important A. flavus virulence factors.     

Purification and characterization of a Bacillus cereus collagenolytic/proteolytic enzyme and its effect on Meloidogyne javanica cuticular proteins

A novel collagenolytic/proteolytic enzyme isolated from the bacterium Bacillus cereus was purified and characterized. The extracellular enzyme was secreted into the growth medium only after induction by collagen. It was purified by two-step chromatography, consisting of gel filtration through a Sephadex G-100 column and then through an anion-exchange column. Molecular mass, as determined by SDS-PAGE was 42.8. The 42.8-kDa collagenase band was eluted from the gel to obtain the purified enzyme. The enzyme was found to have a very wide range of optimal pHs for activity (5.4 – 8.2), and was stable at temperatures between 4 and 40 °C. In addition to its collagenolytic property, the enzyme revealed very strong proteolytic activity, demonstrated by its ability to digest bovine serum albumin. The enzyme's ability to damage nematode cuticles was demonstrated by the digestion of collagens extracted from intact cuticles of second-stage juveniles of the root-knot nematode Meloidogyne javanica.     

Characterization of glutathione S-transferase of the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae): Comparison of its properties of glutathione S-transferases from other lepidopteran insects

An enzyme that possesses the glutathione S-transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS-polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3-10 and at temperatures below 50 °C. The enzyme was also able to conjugate glutathione to 4-hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.     

Studies on glutathione transferase of cowpea storage bruchid, Callosobrochus maculatus F

Insect glutathione transferases have been implicated in insecticides and herbicides metabolism. In this study, glutathione transferase was purified to apparent homogeneity from cowpea storage bruchid (Callosobruchusmaculatus) by anion exchange chromatography of DEAE-Sephacel and affinity chromatography of glutathione-Sepharose 4B. The purified enzyme is slightly acidic, pI 5.5, with a molecular weight of 48.5 ± 4 kDa and is composed of subunit molecular weight of 25 kDa. It exhibited an optimum pH and temperature of 8.0 and 40 °C, respectively. Kinetic data gathered from substrate specificity using 1-chloro-2,4-dintrobenzene,7-chloro-4-nitrobenzene-2-oxa-1,3-diazole, p-nitrophenyl acetate, ethacrynic acid, 1,2-dichloro-4-nitrobenzene and paranitrophenychloride and inhibition studies (cibacron blue, bromosulphophthalein, hematin, oxidized glutathione and S-hexylglutathione) of the enzyme showed that is of sigma class. The GST poorly conjugates 4-hyroxylnonenal, a product of lipid peroxidation, and cumene hydroperoxide and may not be involved in oxidative stress protection. Steady state kinetics and product inhibition studies were consistent with a random sequential detoxification mechanism. The interaction between the GST and the insecticides, Fenvalerate and Cypermethrin, was investigated by inhibition studies, circular dichroism and competitive inhibition spectroscopy. It demonstrated that enzyme was inhibited at a concentration that induced some minor changes in the secondary and tertiary structures of the enzyme consequently decrease the detoxification ability and enzyme stability but might not unfold it.     

Characterization of glutathione S-transferase of the rice leaffolder moth,Cnaphalocrocis medinalis (Lepidoptera: Pyralidae): Comparison of its properties of glutathione S-transferases from other lepidopteran insects

An enzyme that possesses the glutathione S-transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS–polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3–10 and at temperatures below 50 °C. The enzyme was also able to conjugate glutathione to 4-hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.     

α-Amylase inhibitor in local Himalyan collections of Colocasia: Isolation, purification, characterization and selectivity towards α-amylases from various sources

The α-amylase inhibitor from corms of Colocasia collected from Bhota village of Hamirpur district, Himachal Pradesh was purified to 17.21 folds with 61.61% recovery using ammonium sulfate precipitation, gel filtration chromatography (sephadex G-200) and ion exchange chromatography (DEAE-sephadex). A single band of the purified inhibitor was obtained by Native-PAGE. SDS-PAGE revealed the purified inhibitor to be a monomer with molecular weight of 13,900 daltons. The nature of inhibition was found to be of non-competitive type as determined by Lineweaver-Burk plot and a K_i value of 0.54 nmole was obtained by Dixon’s plot. The inhibitor was found to be heat stable and retained 81.50% activity at 70 °C temperature. Inhibitor was found to have pH optima of 6.9. The purified inhibitor was found to have inhibitory activity against α-amylases extracted from the larvae of Callosobruchus chinensis, Tribolium castaneum, Corcyra cephalonica and midgut α-amylase of Spodoptera littoralis. 100% larval mortality of C. cephalonica was observed when fed on wheat flour mixed with 0.0036% (w/w) of purified inhibitor. Purified α-amylase inhibitor was found to inhibit the activity of human salivary α-amylase. It also had inhibitory activity against potato α-amylases and reduced sugar content in treated potato slices. The purified inhibitor was found to be a glycoprotein. In the present study, the ability of the inhibitor to inhibit insect amylases highlights its possible role in pest resistance and post harvest decay of crop plants. Inhibitory activity of α-amylase inhibitor against mammalian amylases could suggest its potential in treatment of diabetes and cure of nutritional problems, which result in obesity.     

Cellulase in the Host–parasite System Phaseolus vulgaris (L.)–Uromyces appendiculatus [Pers.] Link

Activity of cellulase and xylanase in the intercellular washing fluid (IWF) of bean plants (Phaseolus vulgaris, cultivar Fori) was monitored during infection with bean rust (Uromyces appendiculatus). In infected plants, cellulase activity could be detected at 2 days after inoculation and reached its maximum between 7 and 8 days after inoculation. The enzyme activity was not detected in healthy controls. The cellulase had a pH optimum at pH 5.5 and a temperature optimum at 30°C. Complete inactivation of cellulase occurred after heating to 50°C for 30min. In non-denaturing polyacrylamide gradient gels, the enzyme exhibited four bands (molecular masses approximately 70, 95, 120, 170kDa). After isoelectric focusing, eight cellulase isoforms with pI values pI 4.6–4.8; 5–5.1; 5.4; 5.5; 5.9; 6; 6.5; 7 appeared. Two dimensional electrophoresis yielded 13 cellulase isoforms. Unlike cellulase, low levels of xylanase were detected in healthy controls. The activity of this hydrolase did not increase due to rust infection.     

Axenic culture and influence of wetness period and inoculum concentration on infection and development of cercospora blight ofHeliotropium europaeum

Cercospora heliotropii-bocconii is a fungal pathogen of the ephemeral annual weedHeliotropium europaeum. The effects of wetness period and inoculum concentration on disease severity were studied under controlled conditions. The fungus was grown on different artificial culture media and carrot juice agar with 5 g l^–1 yeast extract was found to be the most suitable medium for conidial production under artificial conditions. Abundant disease symptoms only occurred after 8 h of wetness at 20°C. The minimum incubation period before disease symptoms appeared was 8 days following a wetness period of at least 40 h. Inoculum concentration of 1×10⁴ conidia per ml killed plants in less than one month and reduced seed production by two thirds. These results suggest that this pathogen has the potential to reduce plant survival and seed bank replenishment of this annual weed species.     

Partial characterization of a non-proteinaceous suppressor of non-specific elicitors from Cladosporium fulvum (syn. Fulvia fulva)

It is our working hypothesis that suppression of the activity of glycoprotein non-specific elicitors (NSE) from fungal cell walls is required in establishment of basic compatibility in the Cladosporium fulvum-tomato system. A suppressor of NSE-induced necrosis on tomato leaves was partially purified from intercellular fluid (IF) obtained from C. fulvum infected, or uninoculated, leaves. The suppressor was stable to treatment with heat, protease, glucosidase, galactosidase, laminarinase, periodate, and mild acid (0·1 N HCl) and base (0·01 N NaOH). Addition of the chelators, EDTA (15 mM) or EGTA (2 mM), to IF resulted in a marked reduction in suppressor activity. Suppressor activity was partially reduced by dialysis of IF, but activity was lost upon dialysis by prior treatment of IF with urea, or with protease which was then inactivated by heating. Activity of a low molecular weight suppressor, partially purified by dialysis and Sephadex G-25 column chromatography, corresponded with a carbohydrate peak. Pectinase or pectinase-generated oligogalacturonides from polypectate suppressed activity of NSE. However, incubation of NSE with pectinase, followed by heat treatment before assay, did not affect NSE activity. It is suggested that low molecular weight suppressor molecules may originate from action of pectolytic enzymes on host cell walls. In addition to these low molecular weight suppressor, native IF, but not heat treated IF, contained proteins that on in vitro incubation with NSE slowly reduced its ability to induce necrosis.     

Endo-polygalacturonase from Cladosporium cucumerinum elicits lignification in cucumber hypocotyls

The cucumber pathogen Cladosporium cucumerinum produces one endo-polygalacturonase and at least two exo-polygalacturonases during growth in a liquid medium containing citrus pectin as the carbon source. The endo-polygalacturonase was purified nine-fold by ion-exchange chrumatography and gel filtration. The enzyme elicits lignification in cucumber hypocotyls down to a concentration of about 0·08 units ml⁻¹ which corresponds to about 70 ng protein ml⁻¹. It also releases elicitors of lignification from polygalacturonic acid and cucumber cell walls. The enzyme has a pH-optimum between 5·0 and 5·5, a molecular weight of about 38 000 and contains neutral hexose and protein in a ratio of 15:85 (w/w). The endo-polygalacturonase elicits lignification equally effectively in susceptible as in resistant cucumber hypocotyl segments, and releases about the same amount of elicitor from the cell walls of resistant and susceptible cucumber hypocotyls. This does not exclude the involvement of endo-polygalacturonase in the lignification reaction of resistant cucumber plants towards C. cucumerinum, but the specificity of the reaction must apparently be determined by other molecules.     

中华根瘤菌L03几丁质酶纯化及其酶学性质研究

中华根瘤菌Sinorhizobium sp.菌株L03是1株能产生几丁质酶的生防细菌.采用90%饱和度硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose疏水层析等方法获得了菌株L03的几丁质酶.SDS-PAGE检测发现该酶已被纯化,其分子量约为41.3kD.该酶反应的最适温度为45℃;最适反应的溶液pH值为6;酶液在pH5～8条件下和40℃下分别保存1h,酶活力基本保持稳定;Mg2+和Ba2+能显著促进几丁质酶活性上升,而Zn2+、Cu2+和Fe3+等对酶活力有明显的抑制作用.功能分析结果显示,该几丁质酶对供试的几种病原真菌细胞壁都有明显的降解作用.     

淡紫拟青霉几丁质酶的纯化和活性影响因子

 淡紫拟青霉(Paecilomyces lilacinus)是一种重要的植物线虫卵寄生真菌。采用DEAE-22离子交换层析和SephcrylS-300凝胶过滤层析分离纯化到一种淡紫拟青霉胞外几丁质酶,经7.5%聚丙烯酰胺凝胶电脉检测为单一条酶带,Rf值等于0.43。淡紫拟青霉几丁质酶的最适温度为45℃。热稳定性测定表明它在40℃下放置10min可保持原有活力,60℃下10min,完全失活。该酶的最适pH值为5。其pH值稳定性与温度和反应时间有关。20℃、20min,在pH3~9范围内稳定;37℃、1h,则只在pH5~7范围内较稳定。金属离子如Mg²⁺、Ca²⁺、Zn²⁺、Li⁺和Fe²⁺对酶有一定的激活作用,其中Mg²⁺的激活作用最强。相反,Cu²⁺对酶有强烈抑制作用。     

Fusarium solani endo-polygalacturonase from decayed muskmelon fruit: purification and characterization

Fusarium solani is one of the more important fungal pathogens involved in pre- and post-harvest decay of muskmelon fruit. Production of polygalacturonase (PG), by F. solani was studied in vitro and in vivo . The fungus produced at least 14 PG isozymes with pIs of 4.5 to 9.5 in shake culture using pectin as the sole carbon source. When glucose and pectin were used in combination as the carbon source, total PG activity decreased substantially as compared to pectin alone, suggesting that glucose may suppress PG production in vitro . Only one PG isozyme, designated as PG1, was detected in extracts from infected fruit tissue. PG1 from decayed fruit was purified to homogeneity by protein extraction, ultrafiltration, gel filtration chromatography, and cation exchange chromatography. The molecular weight of PG1 was estimated at 38 kDa based on SDS-PAGE with a pI of 9.5 according to IEF-PAGE. PG1 exhibited only endo-PG activity based on viscosity reduction and thin layer chromatography analysis of products released by enzymatic action. The optimum pH for PG1 activity was 6. TheK_m and V_maxof PG1 using polygalacturonic acid as the substrate were 1.34 mg ml^-1and 0.30 unit μg protein^-1, respectively. PG1 effectively macerated fruit tissue which suggests that it may play an important role in decay of muskmelon fruit caused by F. solani .     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

软腐欧氏杆菌的蛋白酶与致病作用的关系

 胡罗卜软腐欧氏杆菌(Erwinia carotovora var.carotovora Dyc)在合成培养基MS中产生两种蛋白酶,但在无细胞滤液中测不到果胶裂解酶和果胶水解酶的活性。用等电聚焦电泳测得这两种蛋白酶都是碱性蛋白酶,等电点分别为8.3和8.9。变性温度为49-50℃,对酸或碱具有较高的稳定性。纯化的蛋白酶单独作用马铃薯块、能使其变软,切片观察表明细胞壁被明显降解。通过滤膜结合法,将供体菌1830/pJB4J1::Tn5的转座子转入到野生型StEcc-12中,从2000个接合子中获得了11个缺少产蛋白酶能力的突变体。这些突变体的分泌性蛋白质等电聚焦图谱与野生型菌株明显不同,其中一个变突体蛋白酶带完全缺失,另外一个则出现8条新的蛋白带,其他5个属部分缺失。这些突变体对马铃薯块组织离析、腐烂的能力都有不同程度的减弱。研究结果认为在由胡罗卜软腐欧氏杆菌引起的软腐病程中蛋白酶和果胶酶是协同作用的,果胶酶降解植物细胞间和胞壁中的果胶质,而蛋白酶则降解植物细胞壁和膜中的蛋白质。     

Eugenol reduces growth and increases activity of S-adenosylmethionine decarboxylase in the phytopathogenic fungusBotrytis fabae

This study was undertaken to examine the possibility that eugenol-induced reductions in growth ofBotrytis fabae are associated with alterations in polyamine metabolism.B. fabae was grown in liquid medium amended with different concentrations of eugenol. Changes in fungal biomass, and activities of enzymes of polyamine biosynthesis and catabolism were studied. An examination was also made of the incorporation of radioactivity from ornithine into polyamines. Activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC) and flux of label from ornithine into the polyamine spermine were greatly increased inB. fabae grown in the presence of eugenol. However, no significant changes were observed in polyamine catabolism or in the concentrations of free polyamines in treated fungal tissue. http://www.phytoparasitica.org posting May 17, 2005.     

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.