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[Preparation of protoplast-like structures of Pseudomonas putida and their reversion to bacterial forms].

A technique is proposed for the preparation of Pseudomonas putida protoplast-like structures by treating the cells with lysozyme in a hypertonic medium containing mono-and divalent cations. Pretreatment of the cells at the logarithmic growth phase with sucrose (0.5 M) for 90 min at the pH 7.9 to 8.0 is a prerequisite for the formation of 'protoplasts'. Under these conditions, the yield of 'protoplasts' reached 99.9% of the total cell number. The protoplast-like structures are capable of reversing into the original bacterial forms in a minimal hypertonic medium containing amino acids which are components of the cell wall. The level of reversion reaches 10% for certain strains.

Bacteriological Techniques↗
PubMed1981

Lectin-bearing protoplasts of Pseudomonas aeruginosa induce capping in human peripheral blood lymphocytes.

The redistribution of surface membrane receptors and cap formation by pseudomonas aeruginosa lectins (Ps-GAL, which binds D-galactose, and Ps-MAN, which binds D-mannose and L-fucose) was studied in human peripheral blood lymphocytes by scanning electron microscopy. When Ps-GAL and Ps-MAN-bearing protoplasts of Pseudomonas aeruginosa were incubated with lymphocytes, cap formation was revealed by the accumulation of protoplasts at one pole of the lymphocyte. No adherence or capping was observed when intact Pseudomonas aeruginosa cells were used. Pretreatment of the lymphocytes by papain or neuraminidase did not enhance the capping. No capping or adherence of protoplasts to lymphocytes was observed in the presence of D-galactose, while addition of D-mannose had no effect. The inhibitory effect of D-galactose on the adherence and capping in human peripheral lymphocytes by Pseudomonas aeruginosa protoplasts suggests that the Ps-GAL lectin is responsible for both phenomena.

Galectins↗
PubMed1995

Morphological aspects of cell wall formation during protoplast regeneration in Candida albicans.

The dynamics of cell wall regeneration in Candida albicans protoplasts was investigated by fluorescence microscopy using a new fluorescent dye, Fungiflora Y, which specifically binds to beta-linked polysaccharides, as well as by electron microscopy. When freshly prepared protoplasts were incubated in osmotically stabilized medium, multiple binding sites of Fungiflora Y were detected on their surface within 15 min of incubation. At this initial stage of reversion, protoplasts began to form microfibrils outside the cytoplasmic membrane. From 1 h onward, the whole cell surface displayed a bright fluorescence. After 3 h of incubation, the reverting protoplasts were covered by an interwoven network of microfibrils. After 5 h of incubation, although most of the revertants had become osmotically resistant, regenerated cell walls in which the constituent materials appeared highly integrated were observed to be still monolayered in profile. Cell wall regeneration was complete after 24 h of incubation, at which time the reverted cells were capable of proliferating.

Candida albicans↗
PubMed1994

Direct transfer of plasmid DNA from intact yeast spheroplasts into plant protoplasts.

We developed a polyethylene glycol (PEG)-mediated direct DNA transfer method from intact Saccharomyces cerevisiae spheroplasts into Arabidopsis thaliana protoplasts. To monitor the DNA transfer from yeast to plant cells, beta-glucuronidase (GUS) reporter gene in which a plant intron was inserted was used as a reporter. This intron-GUS reporter gene on a 2 microns-based plasmid vector was not expressed in yeast transformants, while it expressed GUS activity when the plasmid DNA was introduced into plant cells. When a mixture of 1 x 10(8) of S. cerevisiae spheroplasts harboring the plasmid and 2 x 10(6) of A. thaliana protoplasts was treated with PEG and high pH-high Ca2+ solution (0.4 M mannitol, 50 mM CaCl2, 50 mM glycine-NaOH pH 10.5), GUS activity was detected in the extract of the plant cells after a three-day culture. The GUS activity was higher than that of a reconstitution experiment in which the mixture of 1 x 10(8) of S. cerevisiae spheroplasts which did not carry the reporter gene, 2 x 10(6) of A. thaliana protoplasts and the same amount of the reporter plasmid DNA as that contained in 1 x 10(8) of S. cerevisiae spheroplasts, was treated with PEG and high pH-high Ca2+ solution. Moreover, the GUS gene expression was resistant to micrococcal nuclease treatment before and during PEG treatment. From these results, we concluded that plasmid DNA can be directly transferred from intact yeast spheroplasts to plant protoplasts by a nuclease-resistant process, possibly by the cell fusion.

Arabidopsis↗
PubMed1997

Factors affecting transient expression of vector constructs in wheat protoplasts.

Direct uptake of reporter gene constructs with the bacterial beta-glucuronidase (GUS) gene fused to various promoters was achieved to embryogenic cell suspension culture-derived protoplasts of GK Ságvári winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment. Based on GUS specific activity values, it was found that Mg2+ with PEG at 20% final concentration can significantly increase the transient expression in wheat protoplasts in comparison to the Ca(2+)-containing medium. The optimum incubation time in transformation mixture was 5-10 min at 25 degrees C. Transient GUS expression as detected by spectrofluorimetry was positively correlated with the time elapsed after DNA uptake (with maximum activity at 48 h), and the incubation time in GUS reaction mixture. It was also found that the protoplast culture medium plays an important role in the efficiency, and the treated wheat protoplasts cultured in KM medium showed a higher GUS activity than those kept in GM medium. Among the five plasmid constructs 6-16-fold higher promoter activity has been achieved with pKM794 driven by CaMV 35S promoter plus two enhancer elements than with the other constructs tested.

Gene Expression↗
PubMed1993

Replication of maize streak virus mutants in maize protoplasts: evidence for a movement protein.

We have used protoplasts derived from a maize (Black Mexican Sweet) suspension culture to study the replication of maize streak virus (MSV) wild-type (wt) and mutant DNA genomes. Following inoculation with plasmids containing multimeric copies of MSV, both single-stranded (ss) and double-stranded MSV DNA forms were produced, in proportions relative to those seen in infected plants. The immunodetection of coat protein (PV2) and geminate particles confirmed that the protoplasts were able to support the entire multiplication cycle of MSV and therefore were suitable for the study of MSV gene function. Inoculation of protoplasts with V1 gene mutants which are unable to produce systemic infections in plants resulted in DNA replication and encapsidation indistinguishable from that obtained with wt constructs implicating the protein in virus movement. Computer analysis of the V1 protein (PV1) indicated a potential trans-membrane or membrane-embedded domain. Following inoculation of protoplasts with V2 mutants, ssDNA was not detected; the inability of these mutants to spread in plants may be due to a lack of ssDNA, PV2, or a combination of the two. The requirements for systemic infection of MSV are discussed.

Base Sequence↗
PubMed1995

Transient gene expression of antisense RNA and coat protein-encoding sequences reduced accumulation of cherry leafroll virus in tobacco protoplasts.

Cherry leafroll nepovirus (CLRV) is the causative agent of blackline disease, which results in a fatal necrosis of the graft union of English walnut scion on certain rootstocks. Tobacco suspension cell protoplasts were electroporated with plasmid constructions, bearing or not bearing RNA-derived sequences, and, subsequently, were electroporated with CLRV virions or virion RNA Replication of CLRV in protoplasts was demonstrated by accumulation of both positive- and negative-sense CLRV genomic RNAs 1 and 2, capsid antigen, and virions. Three plasmids were tested for antiviral action. These have inserts that were derived from the coat protein gene, inserted in both orientations, and from the 3' terminal sequence that is nearly identical in RNA 1 and RNA 2, oriented for expression of antisense RNA. Plasmids were introduced into protoplasts 12 hr prior to introducing the virions or virion RNA. CLRV accumulation was reduced significantly by prior electroporation of plasmids intended to express coat protein or 3' antisense RNA, but not by electroporation of plasmid without insert or plasmid with coat protein encoding sequences in the antisense orientation. These results demonstrate the utility of transient expression in a protoplast system for comparing the efficacy of a variety of virus-derived and other sequences for their potential application in virus control strategies.

Capsid↗
PubMed1993

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts.

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2-4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was verified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T0 plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T1 and T2 progenies. Enzyme analyses on T1 progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T0 plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.

Acetyltransferases↗
PubMed1993

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize.

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the beta-glucuronidase (GUS) gene, under the control of the doubled enhancer element (the -208 to -46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to -389 bp from ATG) promoter of wheat, alpha-amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10-20 resistant calli, or GUS-expressing colonies after treatment of 10(6) protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80-90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.

Aminobutyrates↗
PubMed1987

Genetic mapping by means of protoplast fusion in Bacillus subtilis.

A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, "x", to be mapped were fused with polyethylene glycol, and purA+ purB+ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.

Bacillus subtilis↗
PubMed1980

Membranes of the protoplast L-form of Proteus mirabilis.

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysacchartide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. Considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of D-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypeptidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10). which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.

Bacterial Proteins↗
PubMed1990

Induction of a long-lived fusogenic state in viable plant protoplasts permeabilized by electric fields.

Electropermeabilized tobacco mesophyll protoplasts are shown to fuse by creating cell contact several minutes after electropulsation. Electropermeabilization was analysed by measuring calcein uptake. Experiments were performed at low temperature to avoid resealing of protoplast transient permeation structures. These results confirm that the long-lived permeabilized state induced by the electric field is associated to a fusogenic state, under viability conditions. This is indicative that as for mammalian cells, the electric field-induced membrane modifications, which give the permeable state, are such as to decrease the magnitude of the intercellular repulsive forces between plant protoplasts. Such a fusion method may be useful for somatic hybrids production with protoplasts showing morphological and physiological differences.

Cell Fusion↗
PubMed1990

Replication of sonchus yellow net virus in infected protoplasts.

Tobacco (Nicotiana edwardsonii and N. benthamiana) protoplasts infected with the plant rhabdovirus sonchus yellow net virus (SYNV) were found to be suitable for studies of replication. SYNV messenger RNAs could be detected within 2 hr postinoculation (PI), accumulated to a maximum within 24 hr, and subsequently declined to undetectable levels by 60 hr. Plus- and minus-sense genomic RNAs appeared later and were most abundant by 36 hr PI, but the levels decreased by 60 hr. The four major SYNV structural proteins could be detected by Western blot serological analyses by 24 hr PI and were present in highest concentrations between 43 and 60 hr PI. Among various glycosylation inhibitors, only tunicamycin treatment of protoplasts altered viral protein patterns and resulted in synthesis of a glycoprotein with an apparent molecular weight (Mr) 10% smaller than that found in untreated protoplasts. Two specific cleavage products of the nucleocapsid protein estimated to be 21,000 and 37,000 Mr appeared in N. benthamiana-infected protoplasts by 60 hr PI, but in the presence of tunicamycin, the cleavage products were evident by 38 hr. This result suggests that specific cleavage of the N protein is correlated with stress of infected cells due to viral accumulation and/or tunicamycin treatment.

Blotting, Western↗
PubMed2004

Trafficking of the plant potassium inward rectifier KAT1 in guard cell protoplasts of Vicia faba.

Trafficking of K+ inward (Kin+) rectifying channels was analyzed in guard cells of Vicia faba transfected with the Kin+ rectifier from Arabidopsis thaliana KAT1 fused to the green fluorescent protein (GFP). Confocal images and whole-cell patch-clamp measurements confirmed the incorporation of active KAT1 channels into the plasma membrane of transfected guard cell protoplasts. The Kin+ rectifier current density of the plasma membrane was much larger in transfected protoplasts than in wild-type (wt) protoplasts. This shows a coupling between K+ channel synthesis and incorporation of the channel into the plasma membrane. Pressure-driven increase and decrease in surface area led to the incorporation and removal of vesicular membrane carrying active Kin+ rectifier in wt and transfected protoplasts. These vesicular membranes revealed a higher channel density than the plasma membrane, suggesting that Kin+ rectifier remains in clusters during trafficking to and from the plasma membrane. The observed results can be explained by a model illustrating that vesicles of a pre-plasma membrane pool carry K+ channels preferentially in clusters during constitutive and pressure-driven exo- and endocytosis.

Arabidopsis Proteins↗
PubMed1982

Responses of corn root protoplasts to exogenous reduced nicotinamide adenine dinucleotide: Oxygen consumption, ion uptake, and membrane potential.

Addition of 1.5 mM NADH tripled the O(2) consumption in corn root protoplasts. The stimulation was temperature and pH dependent, specific to NADH, and accompanied by a 2- to 3-fold increase in K(+) and P(i) uptake into protoplasts. The increase in ion uptake was not due to the accumulation of NADH into protoplasts. The effect of exogenous NADH on O(2) consumption and ion uptake was also evident in corn root segments but to a lesser extent. A 20-mV hyperpolarization of protoplast membrane potential occurred on addition of NADH and was abolished by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Increases in cell volume of 30% and 40% were detected in response to NADH/H(+) and NADH/H(+)/K(+), respectively. The data are discussed in terms of a transmembrane redox reaction and the possibility that some part of the energy-linked ion transport may be driven by a NADH --> O(2) electron-transport system in the plasmalemma.

Journal Article↗
PubMed1984

Lamellar-to-hexagonalII phase transitions in the plasma membrane of isolated protoplasts after freeze-induced dehydration.

In protoplasts isolated from nonacclimated rye leaves (Secale cereale L. cultivar Puma), cooling to -- 10 degrees C at a rate of 1 degrees C/min results in extensive freeze-induced dehydration (osmotic contraction), and injury is manifested as the loss of osmotic responsiveness during warming. Under these conditions, several changes were observed in the freeze-fracture morphology of the plasma membrane. These included (i) lateral phase separations in the plasma membrane, (ii) aparticulate lamellae lying next to the plasma membrane, and (iii) regions of the plasma membrane and associated lamellae in various stages of lamellar-to-hexagonalII transition. These morphological changes also were observed after equilibration in 5.37 osmolal sorbitol at 0 degrees C, which produced a similar extent of dehydration as did freezing to -- 10 degrees C. In contrast, only small areas of lateral phase separation in the plasma membrane, with no observable aparticulate lamellae or hexagonalII configurations, were observed in protoplasts supercooled to -- 10 degrees C. Therefore, freeze-induced lamellar-to-hexagonalII phase transitions in the plasma membrane are a consequence of dehydration rather than subzero temperature per se. When suspensions of protoplasts isolated from cold-acclimated leaves were frozen to -- 10 degrees C, no injury was incurred, and hexagonalII phase transitions were not observed. No hexagonalII phase was observed even at -- 35 degrees C, though acclimated protoplasts are injured at this temperature.

Cell Membrane↗
PubMed1986

Expression of a foreign gene linked to either a plant-virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat, and sorghum.

The bacterial chloramphenicol acetyltransferase (CAT) gene was expressed in protoplasts of three important graminaceous plant species after introduction of the gene by electroporation. Gene transfer occurred when high-voltage electric pulses were applied either directly or indirectly (without anode contact) to a solution containing plasmid DNA and protoplasts of rice, wheat, or sorghum. The indirect method was more rapid, resulted in higher protoplast viability, and was less subject to contamination than the direct-contact method. Gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the 35S promoter of cauliflower mosaic virus or the copia long terminal repeat promoter of Drosophila. Together with recent advances in regeneration of callus and whole plants from protoplasts, this system makes it possible to study inheritance and expression of genes introduced into graminaceous monocotyledonous plants.

Journal Article↗
PubMed1989

Functional evidence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts.

Tobacco mesophyll protoplasts were previously shown to respond to naphthaleneacetic acid by modifying their transmembrane potential difference. In the present work, evacuolated protoplasts were used to show that this response resides only at the plasmalemma. This electrical response was investigated by using polyclonal antibodies directed against plasma membrane antigens presumably involved in the reception and transduction of the auxin signal. An IgG fraction from an antiserum directed against the membrane auxin-binding protein from maize coleoptile completely inhibited the naphthaleneacetic acid-induced response of tobacco protoplasts. The suppression of the auxin-induced variation in the transmembrane potential difference by an IgG preparation directed against the plasmalemma ATPase from yeast demonstrated the involvement of the ATPase in the electrical response. Variation induced by fusicoccin in the transmembrane potential difference of tobacco protoplasts was unaffected by the anti-auxin-binding protein IgG fraction but was completely suppressed by the anti-ATPase IgG preparation. These results demonstrate the presence of a membrane receptor for auxin at the plasmalemma, the binding of the hormone to this receptor leading to the activation of the proton-pumping ATPase. They also show that at least the primary steps of activation by naphthaleneacetic acid are distinct from those of the fusicoccin-induced response.

Journal Article↗