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At least 973 records · Page 54Linked to original sources
PubMed2026

A streamlined protocol for small-scale protoplast generation and CRISPR/Cpf1-mediated genome editing in Fusarium oxysporum.

Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomic analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR-Cas9 limitations, such as restricted protospacer adjacent motif (PAM) sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using filter column-based methods that greatly reduce enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with shorter guide RNA design and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development.

CRISPR/Cpf1↗
PubMed2004

Auxin induces an increase of Ca2+ concentration in the cytosol of wheat leaf protoplasts.

Auxin addition to protoplasts isolated from leaves of 6-day-old wheat seedlings (Triticum aestivum L. cv. Kadett) induced a rapid increase in the cytosolic calcium concentration [Ca2+]cyt. The shifts in [Ca2+]cyt were detected by use of fluorescence microscopy in single protoplasts loaded with the calcium binding tetra[acetoxymethyl]ester of the fluorescent dye, Fura 2. Addition of the synthetic auxin naphthyl acetic acid, 1-NAA, induced an increase in [Ca2+]cyt within 5-10s, while the physiologically non-active analogue, 2-NAA, did not. The amplitude of calcium increase depended on the concentration of 1-NAA. Since the process was affected by different concentrations of Ca2+ in the external medium, and since the calcium channel blockers (nifedipine and verapamil) postponed and inhibited the reaction, it is suggested that auxin primarily activates Ca2+-permeable channels in the plasma membrane. In the presence of low external calcium concentration (0.1 mM), 5 mM LiCl almost totally blocked the increase in [Ca2+]cyt, indicating a possible involvement of tonoplast Ca2+-channels in the auxin-induced [Ca2+]cyt shift. Thus, calcium signalling induced by auxin involves both external and internal Ca2+ pools.

Calcium↗
PubMed1998

Permeation of redox species through a cell membrane of a single, living algal protoplast studied by microamperometry.

Permeation of several redox species through a cell membrane of a single algal protoplast (radius 100 microns) was investigated by amperometry with a Pt microdisk electrode (disk radius, 6.5 microns) located near the membrane. The redox current observed at the microelectrode decreased as the microelectrode approached the cell membrane since the membrane acted as a barrier for diffusion of redox species from bulk to the microelectrode. Permeability coefficient (Pm) of the protoplast membrane was determined by the quantitative analysis of the variation of the redox current with microelectrode-membrane distance using digital simulation. The Pm values for Fe(CN)6(4-), Fe(CN)6(3-), Co(phen)3(2+), ferrocenyl methanol(FMA) and p-hydroquinone(QH2) were < or = 1.0 x 10(-4), < or = 1.0 x 10(-4), 1.0 x 10(-3), 5.0 x 10(-3) and 2.0 x 10(-2) cm/s, respectively. Using these Pm values, the concentration changes inside a model cell and chloroplast were theoretically calculated.

Cell Membrane Permeability↗
PubMed1992

Interaction of DNA with cationic liposomes: ability of transfecting lentil protoplasts.

The vesicle made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were multilamellar and rather homogeneous in shape as seen by transmission electron microscopy. Upon addition of circular DNA plasmids of different lengths to the liposomes, the formation of vesicle clusters around the DNA filament was observed, with dimensions dictated by the ratio DNA/lipid. These liposomes were able to transfect lentil (Lens culinaris) protoplasts inside the cells two different reporter genes, chloramphenicol-acetyltransferase and beta-glucuronidase. The activity of these two enzymes could be found in the cell lysates after 24 h from the incubation of protoplasts with the lipid-DNA complexes.

Chloramphenicol O-Acetyltransferase↗
PubMed1988

Changes in intracellular redox and energy status during induced transplasma membrane electron transport in Cuscuta protoplasts.

Extracellular reduction of ferricyanide was exhibited by isolated Cuscuta protoplasts. A larger decrease in NADH than NADPH levels of the ferricyanide-treated protoplasts pointed to the major involvement of the former as an electron donor. Glutathione levels were also found to be lowered in similarly treated tissue. The time-dependent variation in intracellular ATP levels in presence of ferricyanide supported the concept of plasma membrane ATPase activation during transplasma membrane electron transport in eukaryotes.

Adenosine Triphosphatases↗
PubMed1969

Passive electrical properties of microorganisms. IV. Studies of the protoplasts of Micrococcus lysodeikticus.

Observations of protoplasts of Micrococcus lysodeikticus show that removal of the cell wall of this organism decreases the dielectric constant by two orders of magnitude. The upper limit of the effective, homogeneous conductivity for the protoplast is 0.001 mho/m as compared with 0.045 mho/m for the intact cell. These results conclusively demonstrate the dominant effect of the cell wall on the low frequency dielectric properties of bacteria.

Cell Wall↗
PubMed1999

Microamperometric measurements of photosynthetic activity in a single algal protoplast.

The effects of p-benzoquinone (BQ) on photosynthetic and respiratory electron transport in a single algal protoplast (radius, 100 microm) was investigated quantitatively by amperometric measurements using microelectrodes. Under light irradiation (25 kLx) in the presence of 1.00 mM BQ, a single protoplast consumed BQ by (2.9 +/- 0.2) x 10(-13) mol/s and generated p-hydroquinone (QH2) by (2.7 +/- 0.3) x 10(-13) mol/s, suggesting that BQ was quantitatively reduced to QH2 via the intracellular photosynthetic electron-transport chain. The generation of QH2 increased with light intensity and with concentration of BQ added to the outside solution but became saturated when the light intensity was above 15 kLx or the BQ concentration was higher than 0.75 mM. The addition of 3-(3, 4-dichlorophenyl)-1,1-dimethylurea, a photosynthetic electron-transport inhibitor, decreased the generation of QH2 upon light irradiation, suggesting that BQ accepts electrons from a site in the photosynthetic electron-transport chain after the photosystem II site. The presence of 1.00 mM BQ increased the generation of photosynthetic oxygen by approximately (2.6 +/- 1.0) x 10(-13) mol/s, which was approximately 1.5-2 times larger than that expected from the consumption of BQ. The electrons produced by the additional generation of oxygen is used to reduce intracellular species as well as to reduce BQ.

Benzoquinones↗
PubMed2000

Cleavage of lamin-like proteins in in vivo and in vitro apoptosis of tobacco protoplasts induced by heat shock.

Apoptosis in heat shock-treated tobacco protoplasts was evidenced by DNA fragmentation, flow cytometric analysis and activation of caspase 3-like protease. Furthermore, an in vitro apoptosis system was established which reproduced the apoptotic events. Western blotting analysis using an antibody against lamin A and C showed that in both in vivo and in vitro systems lamin-like proteins were cleaved into a 35-kDa fragment, and that lamin-like protein degradation precedes DNA fragmentation. Moreover, we found a 22.8-fold increase in caspase 6-like activity in cytosol of heat-treated protoplasts as compared with the control.

Apoptosis↗
PubMed2001

Antibodies to the CFTR modulate the turgor pressure of guard cell protoplasts via slow anion channels.

The plasma membrane guard cell slow anion channel is a key element at the basis of water loss control in plants allowing prolonged osmolite efflux necessary for stomatal closure. This channel has been extensively studied by electrophysiological approaches but its molecular identification is still lacking. Recently, we described that this channel was sharing some similarities with the mammalian ATP-binding cassette protein, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel [Leonhardt, N. et al. (1999) Plant Cell 11, 1141-1151]. Here, using the patch-clamp technique and a bioassay, consisting in the observation of the change in guard cell protoplasts volume, we demonstrated that a functional antibody raised against the mammalian CFTR prevented ABA-induced guard cell protoplasts shrinking and partially inhibited the slow anion current. Moreover, this antibody immunoprecipitated a polypeptide from guard cell protein extracts and immunolabeled stomata in Vicia faba leaf sections. These results indicate that the guard cell slow anion channel is, or is closely controlled by a polypeptide, exhibiting one epitope shared with the mammalian CFTR.

Animals↗
PubMed2001

Molecular identification of phenylalanine ammonia-lyase as a substrate of a specific constitutively active Arabidopsis CDPK expressed in maize protoplasts.

Phenylalanine ammonia-lyase (PAL) is a key enzyme in pathogen defence, stress response and secondary metabolism and is subject to post-translational phosphorylation. In order to address the significance of this phenomenon it is necessary to identify the protein kinase (PK) responsible and place it in its regulatory circuit. Using protoplast transient expression of Arabidopsis kinase genes coupled to immunocomplex kinase assay, it has been possible to screen for specific PAL kinase. We show here that AtCPK1 (calcium dependent PK), but not other closely related PKs could phosphorylate both a recombinant PAL protein and a peptide (SRVAKTRTLTTA) that is a site phosphorylated in vivo. Identification of the specific CDPK as a PAL kinase now opens up the possibility of exploring the calcium link in biotic stress signalling, salicylate and phytoalexin production as well as the significance of PAL phosphorylation. The protoplast transient expression system is a potentially powerful method to determine and screen for plant gene functions utilising genomic and proteomic data.

Amino Acid Sequence↗
PubMed2002

First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts.

Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H(+) ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all 'patch-catch' experiments and could be detected even in 2560-fold dilutions of the pollen contents. H(+) ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT-PCR experiments, a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts.

Lilium↗
PubMed1997

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts.

Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

Actin Cytoskeleton↗
PubMed1999

Influence of additives on the protoplasts electrofusion.

Various neutral or charged surface active substances were used for testing the influence of additives on the electrofusion of barley protoplasts. It was found that neutral surface active agents DX, TAGB, Span-80 and AEO-9 could promote the electrofusion. The positively charged surface active agents Bardac 2080, Bardac 2280 and amphoteric surface active agents dodecyl-propyl betaine and CAB betaine also promote the electrofusion, but at high concentration the electrofusion efficiency will reduce. The negatively charged polymer agents Cibacron blue DX, Fluoresceinylthiocarbamoyl DX, and active surface substances K12 and Carsonol TLS- presented negative effect. These phenomena were discussed from the view of adsorption of additives on the membrane and the interactions between protoplasts.

Cell Fusion↗
PubMed1998

Identification of UV/blue light-response elements in the Arabidopsis thaliana chalcone synthase promoter using a homologous protoplast transient expression system.

To identify DNA sequences of the Arabidopsis thaliana chalcone synthase gene (CHS) concerned with induction by UV-B and UV-A/blue light, AtCHS promoter constructions were assayed by transient expression in protoplasts prepared from two different lines of cultured A. thaliana cells. The protoplasts responded similarly to A. thaliana leaf tissue in light-dependent CHS transcript accumulation. The reporter enzyme beta-glucuronidase (GUS) was used to monitor light-responsive promoter activity. A 1972 bp promoter conferred UV-B and UV-A/blue light induction of GUS activity. Deletion to 164 bp resulted in reduced promoter strength but retention of responsiveness to UV-B and UV-A/blue light. Further deletion abolished transcriptional activity. The 164 bp promoter contains sequences closely resembling LRUPcCHS, (light-responsive unit of the Petroselinum crispum CHS promoter). This A. thaliana CHS promoter region, designated LRUAtCHS, was sufficient to confer UV-B and UV-A/blue light responsiveness to a heterologous core promoter. Mutation of sequences in LRUAtCHS corresponding to the ACGT element and the MYB recognition element of LRUPcCHS resulted in inactivation of the 164 bp and 335 bp promoter deletions. However, the mutant 668 bp promoter retained residual UV-B and UV-A/blue light-induced expression, indicating the presence of additional functional sequences upstream of -335. Mutation of a single G-box-like sequence around -442 had no effect on light responsiveness, indicating that it does not function in light regulation of this promoter. Since no difference in responsiveness to UV-B and UV-A/blue light was observed with any promoter variant, we conclude that the two phototransduction pathways regulate transcription factors which interact with common promoter elements. The results from-our analysis of a A. thaliana light-responsive promoter will facilitate the study of light-dependent gene regulation by genetic means in Arabidopsis thaliana.

Acyltransferases↗
PubMed1967

Studies on the interaction of homologues of spermine with deoxyribonucleic acid and with bacterial protoplasts.

Four homologues of the naturally occurring polyamine spermine, of the type H(2)N.[CH(2)](3).NH.[CH(2)] (n).NH.[CH(2)](3).NH(2) where n=2, 3, 5 and 6, have been synthesized. Their ability to stabilize Escherichia coli protoplasts against osmotic lysis was compared with that of spermine. All homologues were approximately as effective as spermine. The effect of low concentrations of the homologues on the T(m) of calf thymus DNA and of Aerobacter aerogenes DNA in 0.03m-sodium chloride-1mm-potassium dimethylglutarate buffer, pH6.2, was tested. The increase in T(m) for a given concentration of amine was found to be n=5>n=4 and n=6> n=3>n=2. When calf thymus DNA in 0.15m-sodium chloride-15mm-sodium citrate was used spermine gave the highest increase in T(m). It is concluded that the stabilization of E. coli protoplasts by tetra-amines is a non-specific effect independent of chain length, whereas the elevation of T(m) of DNA is a more specific effect which depends on chain length.

Amines↗
PubMed1970

Secretion of cell-wall glycoproteins by yeast protoplasts. Effect of 2-deoxy-D-glucose and cycloheximide.

The effect of 2-deoxy-d-glucose and cycloheximide on the synthesis and secretion of the cell-wall constituents protein and mannan in yeast protoplasts was examined in detail. Although the 2-deoxy-d-glucose hardly influenced protein synthesis, a significant parallel inhibition of carbohydrate and protein secretion into the medium was observed. The mechanism of this inhibition is considered as an interference of metabolites of 2-deoxy-d-glucose with the synthesis of yeast mannan. Cycloheximide, which is an effective inhibitor of protein synthesis in yeast (Kerridge, 1958), inhibited the secretion of non-diffusible carbohydrate in yeast protoplasts, but on the other hand had no effect on the activity of particulate yeast mannan synthetase. Our results clearly show that blocking the synthesis of either part of the mannan-protein complex prevents the extracellular appearance of the other component. The nature of this phenomenon is discussed.

Carbon Isotopes↗
PubMed1977

Chitin biosynthesis in protoplasts and subcellular fractions of Aspergillus fumigatus.

The biosynthesis of chitin has been obtained in broken mycelia and protoplasts of the fungus Aspergillus fumigatus. The specific activity of chitin synthase (EC 2.4.1.16) in a membrane preparation from protoplasts derived from the hyphal tips of A. fumigatus was 26.8-fold greater than that of the chitin synthase in broken mycelia, indicating that the active chitin synthase is located primarily in a membrane-bound site at the hyphal tip. Polyoxin D was a potent competitive inhibitor of the enzyme, having Ki 5.2 +/- 0.8 micron with respect to the natural substrate UDP-N-acetyl-D-glucosamine, which has Km 1.58 mM.

Anti-Bacterial Agents↗