The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Production of laccase by a newly isolated deuteromycete fungus Pestalotiopsis sp. and its decolorization of azo dye.

The effect of various carbon and nitrogen sources on the production of laccase by newly isolated deuteromycete Pestalotiopsis sp. was tested under liquid-state fermentation. Twenty grams per liter of glucose and 10 g l(-1) ammonium tartrate were found to be the optimized concentrations of carbon and nitrogen sources, respectively. The influence of different inducers and inhibitors on the laccase production was also examined. Adding the Cu up to optimum concentration of 2.0 mM in medium (include 20 g l(-1) glucose and 10 g l(-1) ammonium tartrate), the highest laccase activity of 32.7 +/- 1.7 U ml(-l) was achieved. Cu had to be supplemented after 2 days of growth for its maximal effect, an addition after 6 days of growth, during which laccase activity was dominantly formed, resulted in distinctly reduced laccase activity. In addition, Direct Fast Blue B2RL can be effectively decolorized by crude laccase, the decolorization percentage of which was 88.0 +/- 3.2% at pH 4.0 within 12 h. The results suggest that Pestalotiopsis sp. is a high potential producer of the industrially important enzyme laccase.

The toxicity of a crude enzyme extract from Gliomastix murorum in Swiss Albino mice.

A crude enzyme extract from a fungus, Gliomastix murorum, could be used in the synthesis of oligosaccharides that are essential to the food and drug industries. This extract may contaminate such products and lead to serious health problems. An investigation on the possible toxicity and mutagenic effect of the extract from this fungal isolate was carried out in Swiss Albino mice. One hundred and 50% of the crude enzyme extract were injected intraperitoneally into the mice every 2 days for 30 days. Normal saline (0.9%), cultivation medium, and cyclophosphamide (80 mg/kg body weight) were given to the control groups. The results indicated that the white blood cell count, serum creatinine, and uric acid of the treated mice were significantly higher than those of the controls (p<0.05), whereas the serum urea-N was lower. For the micronucleus test, mice treated with the extract, especially the group received 100% crude enzyme extract, showed a higher number of micronuclei in polychromatic erythrocytes, as compared to controls. Nevertheless, the micronucleus values were not as high as those found in mice treated with cyclophosphamide, the mutagenic agent. It can be concluded from the results that crude enzyme extract had minor toxic effects on various organ systems tested; more extensive investigation on the safe use of this extract is therefore necessary.

Formation of bound residues of 8-hydroxybentazon by oxidoreductive catalysts in soil.

This study was performed to determine which oxidoreductive catalysts were most efficient in catalyzing the binding of 8-hydroxybentazon to soil humic substances. 8-Hydroxybentazon was completely transformed by an oxidoreductive enzyme, laccase of Myceliophthora thermophila, at pH 3.0-7.0 within 30 min. When abiotic catalysts, manganese(IV), iron(III), and aluminum oxides were used in the same pH range, 8-hydroxybentazon was completely transformed only by manganese(IV) oxide (delta-MnO2), but a relatively small amount of 8-hydroxybentazon was transformed by iron(III) oxide and aluminum oxide. The adsorption of 8-hydroxybentazon in the soil showed an H-type and coincided well with the Langmuir isotherm. To better understand the factors involved in the rapid and strong binding of 8-hydroxybentazon with soil humic substances, 8-hydroxybentazon transformation by oxidoreductive catalysts was studied in various soil conditions: air-dried, preincubated, sterilized, and iron(III) oxide and manganese(IV) oxide free. 8-Hydroxybentazon was completely transformed within 24 h in the decreasing order of preincubated, air-dried, and sterilized soils. However, little transformation was observed in the iron(III) oxide and manganese(IV) oxide free soils. These results suggest that the major catalyst responsible for the rapid and strong binding of 8-hydroxybentazon to soil humic substances is a metal oxide, manganese(IV) oxide, not a soil oxidoreductive enzyme.

Selection of pectinesterase overproducing fungal strains for the low degree methoxylated pectin preparation.

The selection of the novel pectinesterase (PE) producing strains is an important step in their biotechnological application. The present work displays the purposeful multistage selection of strains with the increased ability to synthesize extracellular PE among 700 filamentous fungal collection cultures belonging to 45 genera and 93 species. A scheme of targeted multistage screening of PE producers has been developed. At the first stage of the screening 23 potentially active strains belonging to the genera Penicillium, Fusarium, Aspergillus, Rhizopus, Gliocladium, etc., were selected. Strains of the Trichoderma, Cochliobolus, Cladosporium and Thielavia genera not yet described as PE-active ones were selected. The P. funiculosum species (0.28-0.56 units/ml) possessed higher ability to synthesize PE. High level of the PE activity was revealed in P. rubrum (0.37 units/ml). No correlation between the level of fungal biomass accumulation on pectin-containing medium and the level of their PE activity was observed. A principal chance of the fungal PE substitution for some chemical reagents at the stage of demethoxylation of the pectin-containing raw materials should be noted. The technology of the fungal PE application for production of the low-degree methoxylated pectin is under development. Such pectin is necessary for the low-calorie jellfying products in the food industry and in medicine as a preventive agent removing the toxic substances from the human body.

Modification of the carbohydrate composition of sulfite pulp by purified and characterized beta-xylanase and beta-xylosidase of Aureobasidium pullulans.

Both beta-xylanase and beta-xylosidase were purified to homogeneity from a xylose-grown culture of Aureobasidium pullulans. Cellular distribution studies of enzyme activities revealed that beta-xylanase was an extracellular enzyme, during both the exponential and stationary phases, whereas beta-xylosidase was mostly periplasmic associated. The beta-xylanase exhibited very high specificity for xylan extracted from Eucalyptus grandis dissolving pulp, whereas the beta-xylosidase was only active on p-nitrophenyl xyloside and xylobiose. Comparison of kcat/Km ratios showed that the beta-xylanase hydrolyzed xylan from dissolving pulp 1.3, 2.1, and 2. 3 times more efficiently than Eucalyptus hemicellulose B, Eucalyptus hemicellulose A, and larchwood xylan, respectively. The beta-xylosidase exhibited a transxylosylation reaction during the hydrolysis of xylobiose. When applied on acid sulfite pulp, both enzymes released xylose and hydrolyzed xylan to a different extent. Although beta-xylosidase (0.4 U/g pulp) liberated more xylose from pulp than beta-xylanase (4.7 U/g pulp), it was responsible for only 3% of xylan solubilization. Treatment of pulp with beta-xylanase liberated 51.7 microgram of xylose/g and hydrolyzed 10% of xylan. The two enzymes acted additively on pulp and removed 12% of pulp xylan. A synergistic effect in terms of release of xylose from pulp was observed when the enzyme mixture of beta-xylanase and beta-xylosidase was supplemented with beta-mannanase. However, this did not result in further enzymatic degradation of pulp xylan. Both beta-xylanase and beta-xylosidase altered the carbohydrate composition of sulfite pulp by increasing the relative cellulose content at the expense of reduced hemicellulose content of pulp.

Production of lipase by soil fungi and partial characterization of lipase from a selected strain (Penicillium wortmanii).

Filamentous fungi from soil were screened for their ability to produce lipase. Among 56 filamentous fungi tested, one strain identified as Penicillium wortmanii was selected as the highest lipase producer. Maximum lipase production (12.5 U/ml) was obtained in 7-days cultures utilizing 5% (w/v) olive oil as the carbon source. Optimum pH and temperature for crude lipase were 7.0 and 45 degrees C, respectively. The enzyme was stable at 40 and 45 degrees C and it retained about 55% of its activity when heated at 50 degrees C for 1 hour.

Cloning, expression, and substrate specificity of MeCPA, a zinc carboxypeptidase that is secreted into infected tissues by the fungal entomopathogen Metarhizium anisopliae.

To date zinc carboxypeptidases have only been found in animals and actinomycete bacteria. A cDNA clone (MeCPA) for a novel fungal (Metarhizium anisopliae) carboxypeptidase (MeCPA) was obtained by using reverse transcription differential display polymerase chain reaction to identify pathogenicity genes. MeCPA resembles pancreatic carboxypeptidases in being synthesized as a precursor species (418 amino acids) containing a large amino-terminal fragment (99 amino acids). The mature (secreted) form of MeCPA shows closest amino acid identity to human carboxypeptidases A1 (35%) and A2 (37%). MeCPA was expressed in an insect cell line yielding an enzyme with dual A1 + A2 specificity for branched aliphatic and aromatic COOH-terminal amino acids. However, in contrast to the very broad spectrum A + B-type bacterial enzymes, MeCPA lacks B-type activity against charged amino acids. This is predictable as key catalytic residues determining the specificity of MeCPA are conserved with those of mammalian A-type carboxypeptidases. Thus, in evolutionary terms the fungal enzyme is an intermediate between the divergence of A and B forms and the differentiation of the A form into A1 and A2 isoforms. Ultrastructural immunocytochemistry of infected host (Manduca sexta) cuticle demonstrated that MeCPA participates with the concurrently produced endoproteases in procuring nutrients; an equivalent function to digestive pancreatic enzymes.

Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties.

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37 degreesC ranged from 23 to 196 U. (mg of protein)-1, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, alpha- and beta-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.

Modelling of simultaneous production of polygalacturonase and exopolysaccharide by Aureobasidium pullulans ATHUM 2915.

The polymorphic fungus Aureobasidium pullulans ATHUM 2915, produced significant quantities of extracellular polygalacturonase and polysaccharide when grown, under controlled conditions, in liquid medium with pectin and glucose as carbon sources and nitrogen source as limited factor. Growth, substrate consumption and products formation were simulated by a structured mathematical model, which was compared with the experimental data from batch culture in a chemostat. This model was applied successfully in the study of some essential parameters influenced the process at various pH values.

Endopolygalacturonase genes from Colletotrichum lindemuthianum: cloning of CLPG2 and comparison of its expression to that of CLPG1 during saprophytic and parasitic growth of the fungus.

Following the previous isolation of CLPG1, a gene encoding an endopolygalacturonase (endoPG) secreted into the culture filtrate of Colletotrichum lindemuthianum, we have isolated and sequenced an additional endoPG gene, CLPG2. This gene is present as a single copy in the genome of the fungus. At the amino acid level, CLPG2 shows 61% identity to CLPG1 and between 37 to 59% identity to other fungal endoPGs. RNA blot analyses of endoPG gene expression were followed with specific probes during in vitro culture of the fungus. When conidia were used to inoculate a synthetic medium containing pectin as sole carbon source, only CLPG1 was found to be expressed after 3 days of culture. However, transferring the mycelium grown on glucose for 4 days to a pectin-containing medium allowed the detection of CLPG1 and CLPG2 transcripts as early as 12 h after transfer on this substrate. Expression of CLPG2 was transient while that of CLPG1 was more prolonged. Immunocytological localization of endoPG in C. lindemuthianum-infected bean tissues with antibodies against CLPG1 confirmed that the protein is produced in planta and is associated with extensive degradation of the host cell wall. Detection of endoPG transcripts by reverse transcription-polymerase chain reaction revealed that CLPG1, but not CLPG2, is expressed at the beginning of the necrotrophic stage of infection. These results show that the two endoPG genes are differentially expressed and that CLPG1 encodes the major secreted endoPG both during saprophytic growth and during plant infection.

Cloning and characterisation of glutamine synthetase from Colletotrichum gloeosporioides and demonstration of elevated expression during pathogenesis on Stylosanthes guianensis.

Experiments were designed to clone and identify genes of the fungal phytopathogen Colletotrichum gloeosporioides expressed at high levels during growth on the compatible host Stylosanthes guianensis when compared with expression in axenic culture. A cDNA clone (pCgGS) that hybridised preferentially to a cDNA probe prepared from infected leaves was isolated by the differential screening of a cDNA library from a nitrogen-starved axenic culture of C. gloeosporioides. The DNA sequence of pCgGS is highly homologous to genes for glutamine synthetase (GS) in other organisms. pCgGS contained all of the conserved regions assigned as catalytic domains in GS enzymes. Comparison with genomic sequences indicated that in C. gloeosporioides the GS gene is present as a single copy with three introns. To our knowledge this is the first report of the cloning of a GS from a filamentous fungus. A second clone (pCgRL1) was also isolated and represented a partial cDNA of the 25s rRNA of C. gloeosporioides. Because pCgRL1 did not hybridise to plant rRNA under high-stringency hybridisation conditions, it was used as a reference to quantify the expression of fungal GS mRNA during pathogenesis in S. guianensis compared to fungal growth in axenic culture. The results indicated that elevated expression of GS occurred during pathogenesis of C. gloeosporioides on S. guianensis, particularly at early stages of infection where expression was about six-times higher than during growth in rich culture media. This work also demonstrates that fungal-specific 25s rRNA fragments, such as pCgRL1, have considerable utility as a reference for quantifying pathogen gene expression in infected plants.

The regulation of the vanadium chloroperoxidase from Curvularia inaequalis.

The effects of carbon and nitrogen source on the regulation of the vanadium chloroperoxidase secreted by the fungus Curnularia inaequalis were investigated. The addition of glucose showed a repressing effect on both the observed messenger RNA level and the measured enzyme activities, whereas the addition of glutamate as nitrogen source and the addition of both glutamate and glycerol had no effect. Addition of vanadate had no effect on the level of mRNA. Eight hundred base pairs of the upstream promoter region of vCPO were sequenced and various features of interest are highlighted. Closer inspection of the mycelium revealed that once secreted, vCPO probably remains tightly associated with the hyphae in two forms, one of which may be a proform of the enzyme. A possible cleavage event at the C-terminus may lower its potential for hyphal association and permit its disassociation into the growth medium. A putative role for the vanadium chloroperoxidase is put forward.

Production of an extracellular trypsin-like protease by the fungal plant pathogen Verticillium dahliae.

The plant pathogenic fungus Verticillium dahliae produced extracellular alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a single step, using cation-exchange chromatography, from the filtrate of cultures grown with skim milk as a protein source. N-terminal amino acid sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like protein. The purified enzyme hydrolyzed the synthetic substrate N alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the activity on BAPNA was inhibited by leupeptin, further verifying the trypsin-like nature of the enzyme.

Characterization and cloning of cutinase from Ascochyta rabiei.

Ascochyta rabiei, the causal agent of Ascochyta blight on chickpea plants, secretes a cutinase in the culture filtrate when it is induced by cutin or hydroxylated fatty acids. This cutinase is the main esterase in the culture fluids. The enzyme was purified to homogeneity by three successive chromatographic steps. It showed an apparent molecular weight of 22 kD in SDS-PAGE and cleaved ester bonds of 3H-labelled cutin or p-nitrophenylbutyrate with maximal activities around pH 8. As a serine esterase, cutinase is strongly inhibited by organophosphorous compounds and the most effective inhibitor 2,3,5-trichloropyridine-6-(O-methyl-O-n-butyl)-phosphateester++ + (MAT 9564) shows a Ki value of 0.8 nM. The cutinase gene was cloned from a genomic cosmid library by screening with two oligonucleotides directed against cutinase consensus peptides. The gene was subcloned to a 1.7 Kb SaII/HindIII-insert and sequenced. The cutinase gene codes for a 223 amino acid protein with strong homology to other fungal cutinase sequences. The purified cutinase is encoded by a single copy gene.

Changes in phenol oxidase and peroxidase levels in cocoyam tubers of different postharvest ages infected by Sclerotium rolfsii sacc.

Crude aqueous extracts from the peripheral rot zone of cocoyam tubers infected by Sclerotium rolfsii sacc were shown to be inhibitory to dialysed in vivo polygalacturonase (PG) of the pathogen. The PG inhibitory action, phenol oxidase and peroxidase activities were higher in cocoyam tubers of the Xanthosoma sagittifolium varieties than in those of the Coolocasia esculenta varieties. The levels of phenol oxidase, peroxidase and PG inhibitory activities also decreased as the postharvest age of the tubers increased.

Elicitors and suppressors of hydroxyproline-rich glycoprotein accumulation are solubilized from plant cell walls by endopolygalacturonase.

Treatment of bean cell walls with a pure endopolygalacturonase of the bean pathogen Colletotrichum lindemuthianum race beta released oligogalacturonides and pectic fragments which were separated according to their charge and size. Among galacturonic-acid-containing components, elicitors and suppressors of the plant cell wall hydroxyproline-rich glycoprotein (HRGP) were recovered. Two active small oligogalacturonides with degrees of polymerization of 2 and 3 were characterized by high-performance anion-exchange-chromatography pulsed amperometric detection and fast-atom-bombardment mass spectrometry; they elicited 40-70% hydroxyproline increase within 48 hours at 450 nmol/bean cutting. In contrast, pectic fragments of higher molecular mass, predominantly composed of galacturonic acid and containing sugars typical of the rhamnogalacturonan II domain of pectic polysaccharides, had the ability to substantially suppress hydroxyproline deposition. Maximum suppressor activity, 30-40% below the activity of the control, occurred in 48 hours. In view of the low one-cycle turnover of these proteins in the cell wall and of their structural role, these changes might significantly affect cell wall properties. Elicitation and/or suppression of hydroxyproline were correlated to modifications of HRGP-extensin gene expression. Northern-blot analysis of RNA showed that changes in the transcript intensity became clearly visible within the first 12 hours after the start of either treatment. The results show that pectic components of the plant extracellular matrix have the potential to regulate wall matrix biogenesis. Implications of this finding in plant defense and development are discussed.

Structure and expression of an inhibitor of fungal polygalacturonases from tomato.

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.

Polygalacturonase isozymes from Botrytis cinerea grown on apple pectin.

Five isozymes (four acidic and one basic) of polygalacturonase were separated by chromatofocusing from the culture filtrate of Botrytis cinerea grown on apple pectin. The isozymes, designated as Polygalacturonase I to V, have isoelectric points of 9.7, 4.9, 4.6, 3.7, and 2.7, respectively, with Polygalacturonase III exhibiting the highest specific activity. Polygalacturonase I appeared to function as an endo-polygalacturonase while the other four isozymes act as exo-polygalacturonases. The pH optima of the isozymes range from pH 4.5 to 5.5 with Polygalacturonase V being less sensitive to higher pH compared with the rest of the isozymes.

Kinetic studies of the polygalacturonase enzyme from Colletotrichum lindemuthianum.

The intrinsic protein fluorescence of the polygalacturonase from Colletotrichium lindemuthianum was exploited in stopped-flow experiments aimed at elucidating the kinetic mechanism for this enzyme. Binding of the polymeric substrate polygalacturonic acid (PGA) essentially produced a triphasic fluorescence profile. There was an initial rapid quench in fluorescence, consistent with the rapid formation of the enzyme-substrate complex, with an equilibrium constant of about 8 x 10(-4)% (w/v) PGA (about 0.27 microM). There then followed a near-constant fluorescence phase, attributable to turnover of the enzyme-substrate complex as a steady-state intermediate. As the concentration of the steady-state intermediate became depleted, towards the end of the reaction, there was a partial return of the fluorescence intensity. This phase is attributed to a final, single turnover of the enzyme at the end of the reaction. The fluorescence intensity does not return to its original level due to product remaining bound at the end of the reaction.