Transportation mechanism for vanillin uptake through fungal plasma membrane

Protoplasts of the basidiomycete, Fomitopsis palustris (formerly Tyromyces palustris), were utilized to study a function of the fungal plasma membrane. Fungal protoplasts exhibited metabolic activities as seen with intact mycelial cells. Furthermore, the uptake of certain compounds into the protoplast cells was quantitatively observed by using non-radioactive compounds. Vanillin was converted to vanillyl alcohol and vanillic acid as major products and to protocatechuic acid and 1,2,4-trihydroxybenzene as trace products by protoplasts prepared from F. palustris. Extracellular culture medium showed no activity responsible for the redox reactions of vanillin. Only vanillic acid was detected in the intracellular fraction of protoplasts. However, the addition of disulfiram, an aldehyde dehydrogenase inhibitor, caused an intracellular accumulation of vanillin, strongly suggesting that vanillin is taken up by the cell, followed by oxidation to vanillic acid. The addition of carbonylcyanide m-chlorophenylhydrazone, which dissipates the pH gradient across the plasma membrane, inhibited the uptake of either vanillin or vanillic acid into the cell. Thus, the fungus seems to possess transporter devices for both vanillin and vanillic acid for their uptake. Since vanillyl alcohol was only observed extracellularly, the reduction of vanillin was thought to be catalyzed by a membrane system.     

Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.     

凤尾菇和裂褶菌原生质体非对称融合研究

具有双重营养缺陷型的裂褶菌单核体突变菌株的原生质体经过灭活处理后作为供体与同样带有营养缺陷型标记的风尾菇单核体原生质体融合,得到大量生长速度和菌落形态差异较大的多核体和双核体融合子．这些融合子主要显示了供体菌株的特性,数次转接后,有的融合子从多核体转变成双核体,有的融合子完全恢复了供体亲本双核体的遗传特性,同时获得了一些有意义值得继续探讨的新的生理特性,如菌丝体在木屑 皮以及废棉培养料上生长速度较快,在平板培养基和木屑培养料上均较早较快地形成原基和子实体等．结果表明原生质体非对称融合是一种效率较高的融合方式,可以作为食用菌育种的一种有效途经．     

Transfer of functional adenovirus E1A transcription activator proteins into mammalian cells by protoplast fusion

Human adenovirus 2/5 E1A proteins were used to evaluate protoplast fusion as a method of transferring functional proteins into mammalian cells. Both the E1A 13 and 12 S mRNA products expressed in Escherichia coli are shown to activate in trans adenovirus gene expression following transfer into monkey kidney cells by protoplast fusion. Approximately 20% of the recipient mammalian cells exhibited positive nuclear E1A-specific immunofluorescence following fusion with protoplasts containing E1A protein. E. coli-expressed E1A protein was modified post-translationally in Vero cells following protoplast fusion, as evidenced by its shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. These results establish protoplast fusion as a simple rapid method for examining the functional activity, intracellular distribution, and post-translational modification of E. coli-expressed proteins in intact mammalian cells.     

Effects of non-ionogenic surface active compounds on yeast protoplasts]

The lytic effect of non-ionogenic surface active compounds (SAC), based on polyoxyethylated fatty acids and alkylphenols on the yeast protoplast cytoplasmic membranes and the extracting ability of the SAC with respect to intracellular proteins of intact yeast cells, were studied. It was shown that the lytic activity of the SAC under study depends on the overall effect of the size of their hydrophobic and hydrophylic fragments rather than on the level of the hydrophylic-lypolytic equilibrium of SAC. The absence of correlation between the lytic activity and the extracting ability of SAC is accounted for by the differences in the mechanisms of membrane degradation under the action of SAC on the protoplasts and intact cells. The data obtained support the previously made assumption that the correlation between the size of SAC mycelles and that of the cell wall pores is the limiting factor of the SAC induced protein extraction from the intact cells.     

Glycolipid intermembrane transfer is accelerated by HET-C2, a filamentous fungus gene product involved in the cell-cell incompatibility response

Among filamentous fungi capable of mycelial growth, het genes play crucial roles by regulating heterokaryon formation between different individuals. When fusion occurs between fungal mycelia that differ genetically at their het loci, the resulting heterokaryotic cells are quickly destroyed. It is unclear how het gene products of Podospora anserina trigger heterokaryon incompatibility. One unexplored possibility is that glycosphingolipids play a role because the het-c2 gene encodes a protein that displays 32% sequence identity and an additional 30% similarity to the mammalian glycolipid transfer protein. Here, P. anserina protoplasts containing wild-type het-c2 genes were shown to have greater glycosphingolipid transfer activity than protoplasts with disrupted het-c2 genes, a condition previously linked to altered cell compatibility following hyphal fusion. The observed glycolipid transfer activity could not be accounted for by nonspecific lipid transfer protein activity. Direct assessment showed that purified, recombinant HET-C2 accelerates the intermembrane transfer of glycolipid in vitro, but that the HET-C2 activity is mitigated much less by negatively charged membranes than the mammalian glycolipid transfer protein. The findings are discussed within the context of HET-C2 being a member of an emerging family of ancestral sphingolipid transfer proteins that play important roles in cell proliferation and accelerated death.     

Protoplast formation from selected species of ectomycorrhizal fungi

Summary Conditions for the formation of protoplasts from selected species of ectomycorrhizal fungi are described. The age of the fungal culture and extent of incubation in a lytic enzyme mixture are critical factors for efficient formation of protoplasts. There is a correlation between the distribution of nuclei in hyphal fragments and protoplasts and the frequency of protoplast regeneration. Protoplasts from at least two of the species studied are formed in sufficient numbers and regenerate at suitable frequencies to be useful for development of genetic transformation and cell fusion systems. These fungi can now be considered in experiments designed for the improvement of ectomycorrhizal associations through genetic manipulation of the fungal component.Dedicated to Professor Dr. Dr. hc. K. Esser on the occasion of his 65th birthdayThis research was supported in part by the McIntire-Stennis Cooperative Forestry Research Program and is published as Alabama Agriculture Experiment Station Journal No. 6-881863P     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

Visualisation of transgene expression at the single protoplast level

Protoplasts are currently used to study the expression of genes following transformation. Expression is followed on a population of protoplasts after total protein extraction by conventional western blotting or measure of the enzymatic activity of the transgenic protein. We describe here a new method, called protoplast printing, allowing easy detection of the fraction of cells expressing a certain protein within a population of protoplasts. It consists of immobilization of the protoplast proteins on a nitrocellulose filter, so as to retain the outlines of the cell, followed by immunological detection of the protein of interest. The only special requirement is an antibody specific for the protein. We have studied the expression of the BNYVV coat protein after electroporation of Chenopodium quinoa protoplasts with viral RNAs, and the expression of the NPT II gene in protoplasts isolated from transgenic tobacco plants as well as after direct transfer of plasmid DNA into tobacco protoplasts. In both cases — infection with viral RNAs and transformation with plasmid DNA — expressing and non-expressing cells can be distinguished as early as 12h after transfer of the transgenes.Abbreviations BCIP 5-bromo-4-chloro-3-indolylphosphate - BNYVV beet necrotic yellow vein virus - CaMV Cauliflower Mosaic Virus - NBT nitroblue tetrazolium chloride - NPT II Neomycin phospho transferase     

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.     

凤尾菇和香菇原生质体非对称融合

The protoplasts of auxotrophic mutant monokaryon of Pleurotus. Sajor-caju was used as recipient parenL the protoplasts of wild dikareon of Lentinus edodes were heat-inactivated and used as nuclear donor parent. The fusants of two parents showed some their individual characteristics and physiological difference between themselves. Thrugh protoplast re-isolation and reversion of one randomly selective fusant, two parents had been recovered and the donor parent achieved some new physiological characteristics su…     

Synthesis of heat-shock proteins in Saccharomyces cerevisiae protoplasts

The effect of cellular capsule elimination in Saccharomyces cerevisiae yeasts (protoplast formation) on the heat-shock protein synthesis and the synthesis of the proteins in protoplasts were studied. The methods of mono- and dimeric electrophoresis have demonstrated that (1) about 18 heat-shock proteins with the molecular masses 26-98 Kd are synthesized in cells at 41 degrees C; (2) protoplast formation per se does not induce the synthesis of heat-shock proteins, but the induction of these proteins in protoplasts at 41 degrees C is similar to the one in intact cells. The protoplast formation induces the synthesis of specific proteins different from heat-shock proteins and the synthesis is inhibited by the heat-shock. The heat-shock induces modification of 88 and 86 Kd heat-shock proteins. It inhibits the synthesis of a number of peptides (15-50 Kd) in cells and protoplasts.     

Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhomiae

Preparation and regeneration of mycelial protoplasts from Alternaria eichhorniae were examined. A commercially available muralytic enzyme, Novozym 234, was used for isolation of protoplasts. The mycelial age and the pH of the stabilized buffer affected the formation of protoplasts. The maximum production of protoplasts (3,9 × 10⁸/g fresh weight mycelia) was obtained from 24-h-old mycelia digested with Novozym 234 (20 mg/ml) in a stabilized buffer of pH 6.4 and incubated in the dark at 30°C on a rotary shaker (90 r.p.m.) for 6 h. Morphological characteristics of the protoplasts varied and depended on the age of the mycelia used in protoplast production. Moreover, mycelial age had a highly significant influence (P = 0.0001) on the frequency of protoplast regeneration.     

Degradation of fungal cell walls taking into consideration the polysaccharide composition

Differences in polysaccharide composition of various fungal cell walls were indicated by their susceptibility to enzymatic digestion. This information was used to optimize the enzymatic extraction of intracellular enzymes or the preparation of fungal protoplasts in high yield. Bacterial glucanase and chitinase specially purified were used for this study. Mycelium of Aspergillus niger grown on uric acid was treated with mixtures of glucanase and chitinase. Cell wall breakdown products were analysed and the ratio of chitin to glucan was estimated to be 1:1.4. A. niger protoplast formation was optimized using this information. When the mixture of chitinase to glucanase was 1:1.4, similar to the fungal cell wall composition, a 95% yield of protoplasts was obtained after 30 min and their mean size was 7 μm. However, a ratio of 1.5 to 1 (chitinase to glucanase) was needed for the maximum extraction of uricase. Yield was 10.5 μ g⁻¹cells after 1.5 h incubation at 28°C. Glucanase alone resulted in a maximum yield of 1.9 μ g⁻¹while chitinase alone yielded 6.0 μ g⁻¹under the same conditions.     

The formation of protoplasts from Beauveria bassiana

Summary Beauveria bassiana protoplast formation from blastospores, conidia and mycelia was studied. The method of protoplast formation involves preincubation of the fungal cells with dithiothreitol and subsequent treatment with an enzyme mixture consisting of: cellulase, chitinase, -glucuronidase and lysozyme. Using this procedure protoplasts were formed from blastospores and mycelia but not conidia. Formation of protoplasts from 24 hour old mycelia was 100% efficient using the above conditions. A number of ionic and osmotic protoplast stabilizing agents were tested. Ammonium sulfate was shown to be the stabilizer of choice. Protoplasts were stable when stored at 4° C with a loss of only 17% in 6 days. We suggest that this procedure of protoplast production will allow a gentler method for the extraction and isolation of intact high molecular weight DNA from B. bassiana.     

Mutant strains ofStreptomyces cinnamonensis protoplasts

Mycelium of Streptomyces cinnamonensis mutant strains cultivated in a synthetic medium with glycine produced protoplasts after lysis of cell walls with lysozyme. The protoplast yield was up to 95%. The protoplasts could revert and mycelial forms were thus regenerated. In a sucrose-containing medium the protoplasts stored at 4 degrees C were stable for 2 d.     

Protoplasts of Pyricularia oryzae P2 : Preparation and Regeneration into Hyphal Form

Protoplasts of Pyricularia oryzae P₂, a rice blast mold, were prepared in high yield from the young mycelium of the fungus using lytic enzymes from Bacillus circulans WL 12. The majority of the protoplasts had one nucleus per cell. The protoplasts formed a cell wall and eventually reverted to normal mycelial form in liquid medium. The process of regeneration was studied under phase-contrast and electron microscopes. The protoplast built a very thick wall prior to the protrusion of a germ-tube like hypha. Golgi apparatus-like structures appeared in the early stage of regeneration and disappeared later. Electron-transparent amorphous structures accumulated during regeneration. Lomasomes were observed in the regenerated cell walls.     

水稻草矮病毒在水稻原生质体中的表达

通过建立水稻原生质体培养体系,经多聚鸟氨酸（PLO）介导将提纯的水稻草矮病毒（Rice grassy stunt virus,RGSV）接种到水稻原生质体内,利用酶联免疫吸附法（ELISA）及蛋白免疫印迹法（Western blot）,研究RGSV在水稻原生质体内的生长周期及其编码蛋白的表达情况。结果表明： RGSV在接种后24h左右开始在原生质体内复制,36h左右达到最大值。NS6在15h左右开始表达,在30h左右达到最大值。     

Fractional protein makeup of Candida utilis yeast protoplasts in the process of their growth and development

Candida utilis IBFMY-405 was grown in a synthetic medium with glucose. Cells taken at the logarithmic phase of growth were studied. The cells were treated with the enzyme from Helix pomatia to prepare protoplasts which were separated by differential centrifugation into groups according to their size. Three protein fractions were isolated from each group and the amino acid composition of the proteins was determined. Proteins of the first fraction (cytoplasmic) prevailed in all of the protoplast groups while the content of proteins of the second fraction (intermediate or myosin-like) was the lowest. As the size of protoplasts increased, difference in the quantitative content of proteins from the first and second fractions became less pronounced. The content of proteins of the third fraction was 3.6 and 2.4 times higher in the protoplasts of the medium size than in the largest protoplasts. The amino acid composition of each protein fraction differed quantitatively and qualitatively in all of the protoplast groups.     

Lysis of yeast protoplasts under sodium alkyl sulfates treatment]

The lytic action of homologous series of sodium alkyl sulfates on yeast protoplasts was studied. The concentration dependences study allowed to estimate the lytic concentrations C50 of the agents required for the 50% lysis of protoplasts in the suspension. The data concerning the agents micelle formation in the lytic medium allowed to make some suggestions and to produce a model of the lytic action of alkyl sulfates on the plasma membrane of the protoplasts. The amounts of the agents absorbed on the membrane and involved in the interaction followed by the membrane breakdown in the model are evaluated. On the basis of the data obtained it is concluded that sodium dodecyl sulfate displays the highest lytic activity to the yeast protoplast plasma membrane as compared to the other alkyl sulfates used in the study. The results obtained are discussed in terms of the effect of yeast cell wall on the extraction of intracellular proteins from intact yeast cell under sodium alkyl sulfates action.