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PubMed2005

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune.

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.

Amino Acid Sequence↗

PubMed2007

The use of protoplasts to study innate immune responses.

The use of plant protoplast transient expression system has facilitated the discovery and dissection of many signal transduction pathways in response to hormones, metabolites, and stresses. Recently, Arabidopsis protoplasts also have been used successfully to study plant innate immune responses triggered by pathogen-derived elicitors. Here, we describe the detailed protocols for studying innate immune responses, including cell death and early defense gene regulation activated by two types of elicitors, pathogen-associated molecular patterns and bacterial type III effectors in Arabidopsis protoplasts. This cell-based system simplifies the complex pathogen-plant interactions to pure individual signals and synchronized cell-autonomous responses. The application of this novel approach provides high temporal and spatial resolution to enhance our understanding of the distinct and overlapping signaling events in pathogen-associated molecular pattern- and bacterial type III effector-activated immune responses at the molecular and cellular level.

Arabidopsis↗

PubMed2006

Transfection assays with protoplasts containing integrated reporter genes.

Transient expression assays with protoplasts that utilize stably integrated reporter genes along with transfected effector genes provide several advantages over assays in which both the reporter gene and effector gene(s) are transfected into protoplasts. A protocol for carrying out transient expression assays with Arabidopsis leaf mesophyll protoplasts containing single-copy integrated reporter genes is described.

Arabidopsis↗

PubMed2002

New insights into photosynthetic oscillations revealed by two-dimensional microscopic measurements of chlorophyll fluorescence kinetics in intact leaves and isolated protoplasts.

Chlorophyll fluorescence kinetic microscopy was used to analyze photosynthetic oscillations in individual cells of leaves and in isolated leaf cell protoplasts. Four Brassicaceae species were used: Arabidopsis halleri (L.) O'Kane & Al-Shehbaz, Thlaspi fendleri (Nels.) Hitchc, Thlaspi caerulescens J.&C. Presl and Thlaspi ochroleucum Boiss et Helder. With the latter two, the measurements were extended also to isolated protoplasts. The oscillations were induced under the microscope by exposing dark-adapted samples to actinic irradiance. Detailed analysis of the induced transients revealed that they consist of several processes oscillating with different frequencies and not only one component as reported earlier. Furthermore, it was found that most of these processes are controlled inside each individual cell. This was shown by differences in oscillations in neighboring cells and protoplasts that share a uniform intercellular environment. The frequency of the dominant oscillation frequency depended neither on irradiance nor on CO2 concentration and is, therefore, not controlled by the photosynthetic rate. Characteristic differences in the frequency spectrum and damping of oscillations have been found among the plant species examined.

Brassicaceae↗

PubMed1994

Protoplasts from Phaseolus coccineus L. pulvinar motor cells show circadian volume oscillations.

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.

Circadian Rhythm↗

PubMed1983

Improvement of macrolide antibiotic-producing streptomycete strains by the regeneration of protoplasts.

Three macrolide antibiotic-producing strains, the spiramycin producer Streptomyces ambofaciens, the tylosin producer Streptomyces fradiae, and the cirramycin producer Streptomyces cirratus easily formed protoplasts when treated with lysozyme in hypertonic medium. Each type of protoplast was regenerated to a mycelial form at a frequency of 90 to 100% in regeneration medium supplemented with a plasma expander. In the spiramycin producer S. ambofaciens and the tylosin producer S. fradiae, antibiotic productivities of the regenerated progeny were drastically changed. Some regenerated progeny from the three strains gave higher production. One from S. ambofaciens showed about twice the productivity of the original strain and one obtained after two rounds of protoplast regeneration from S. fradiae produced about three times as much as the original strain.

Anti-Bacterial Agents↗

PubMed2000

[Improved protoplast-derived plants of Astragalus adsurgens through somatic embryogenesis].

Embryogenic callus was obtained only from hypocotyl explants of Astragalus adsurgens and light inhibited the formation of embryogenic callus. A high yield (1.2 x 10(6)/g F. Wt.) of protoplasts with high viability (over 80%) could be isolated from 10-day-old embryogenic callus. Protoplasts were induced to undergo sustained division and to form cell colonies when cultured in agarose-solidified medium (KMP) containing 1/4 strength of mineral salts and supplemented with 1.5 mg/L 2, 4-D, 0.5 mg/L BA and 0.5 mol/L glucose at a plating density of 1.0 x 10(5)mL, where the plating efficinency was 16.8%. Conditioning medium significantly improved the formation of cell colonies. When protoplast-derived colonies were maintained at 4 degrees C for 2 weeks and subsequently transferred onto medium (MS) with 0.1 mg/L NAA and 1.0 mg/L BA, somatic embryogenesis occurred. Frequency of cell colonies producing somatic embryos reached 70%, and the number of somatic embryos per gram cells was over 200. Cultured on hormone-free half-strength MS medium, somatic embryos developed into healthy plantlets with normal chromosome complement.

Cells, Cultured↗

PubMedDate not supplied

[Protoplast production from Candida utilis].

The protoplasts of Candida utilis 295 t were produced with the aid of the lytic enzyme from Helix pomatia. If the cell wall of C. utilis 295 t is not treated with SH-compounds (the best effect was found with L-cysteine), it is resistant to the action of the enzyme. The yield of the protoplasts was 100 per cent after 15 minutes of the incubation with the lytic enzyme if the cells were preliminarily treated with L-cysteine. Optimal conditions for the production of the protoplasts are described.

Candida↗

PubMed2003

[A method for preparation of synaptonemal complexes of meiotic chromosomes from basidial protoplasts of the common mushroom Agaricus bisporus (Lange) Imbach].

For the first time, preparations of synaptonemal complexes (SCs) were made from meiotic chromosomes of white button mushroom (Agaricus bisporus) basidia. It is the first experience of obtaining SC preparations of filamentous fungi from isolated meiosporangium protoplasts. Previously, only yeast SC preparations were obtained following this approach. The method includes four major stages: isolation of basidium protoplasts by treatment of basidia with lytic enzymes, spreading of protoplast nuclei on a filmy support by osmotic shock, staining the preparations with silver nitrate, and examination under light and electron microscopes. The structures of spread premeiotic nuclei, axial elements of chromosomes, SCs, chromatin, and nucleoli were studied at the leptotene-diplotene stage of meiotic prophase I.

Agaricus↗

PubMed1992

[Preparation and characteristics of protoplasts from various strains of Streptomyces roseoflavus var. roseofungini].

It was shown that the variants of the roseofungin-producing organism, which differ in their differentiation, antibiotic activity, structure and cell wall composition had different sensitivity to the protoplasting factors. The protoplasting increased the population heterogeneity: in strain 1128 it was evident from an increased frequency of the secondary colonies, in variant 1-68, folding of the colonies increased and their consistency become milder, sectorial colonies and colonies with coremia formed. It was in principle possible to transform the protoplasts of S. roseoflavus var. roseofungini by plasmid DNA, which suggests that the roseofungin-producing culture may be useful in genetic engineering.

Antifungal Agents↗

PubMed2003

[Protoplast isolation and transformation of Exserohilum turcicum].

Northern corn leaf blight, caused by the fungus Exserohirum turcicum Pass. (Leonard and Suggs), is one of the major diseases in most corn-growing areas of the world. Research on gene tagging of E. turcicum has been limited due to the lack of an efficient transformation system. Since E. turcicum produces and accumulates melamin in cell walls during vegetative growth, it is difficult to efficiently isolate its protoplast. To isolate the protoplast of this pathogen with a high frequency, the effects of cell wall degradation enzymes, including beta-1,3-glucanase (Fungase, Funcelase, Novozyme and Glucanex) and beta-glucuronidase (Driselase, Uskizyme and Kitalase), enzyme concentrations, combinations, strains and medium on the isolation frequency were tested. The isolation frequencies were high enough for transformation when the combinations of (Kitalase + Glucanex + Driselase), (Kitalase + Glucanex) or (Kitalase + Uskizyme) were used. Moreover, the isolation frequencies of protoplast were significantly affected by the cultural morphologies of strain and the growth stage of mycelia. Among the plasmids tested, only plasmid pAN71 is efficient for transformation of E. turcicum. This result will provide some useful information for gene tagging of E. turcicum and other species in Exserohirum.

Ascomycota↗

PubMed1992

Plant protoplasts as genetic tool: selectable markers for developmental studies.

Protoplasts have usually been presented as a methodological tool. Even as such, they make possible an impressive array of applications in plant biology. Here we report on the use of protoplast-derived selectable markers in the study of several disturbed genetic systems with obvious effects on plant development: (1) auxotrophic mutants and the control of amino acid biosynthesis and transport in vegetative and reproductive tissues; (2) introgression of alien genetic information across phylogenetic boundaries by protoplast fusion, a consequence of controlled dedifferentiation-redifferentiation processes and attenuated incompatibility reactions in cultured cells; (3) expression (in)stability of foreign genes in transgenic plants during successive meiotic generations and in crosses between independent transformants.

Cells, Cultured↗

PubMed1991

Standardized isolation of protoplasts.

Isolation of leaf mesophyll protoplasts from tobacco (Nicotiana tabacum) is facilitated using a specially designed digestion chamber. The chamber's airtight seals and various ports reduce the complexity, time and contamination risks which may be associated with standard isolations. The polished glass viewing window allows continuous monitoring of protoplast release, thus facilitating more precise determinations of the optimal digestion time. In concert with a simplified centrifugation step, the protoplast isolation procedure is greatly standardized.

Cell Separation↗

PubMed1990

Size-dependent regeneration of Gibberella protoplasts.

Regeneration ratios of protoplasts formed from the plant pathogenic gibberella fujikuroi with a mixture of lytic enzymes were studied. The heterogeneous population of protoplasts was separated into groups differing in regeneration ratio. The frequency of regeneration was higher for large protoplasts containing an increased number of nuclei.

Cell Nucleus↗

PubMed1990

Plant regeneration from protoplasts of a super Chinese rice (Oryza sativa L.) cultivar--Zhonghua NO.8.

Green rice plants were regenerated from protoplasts, which were derived from cell suspension of Oryza sativa L. cut. Zhonghua No. 8, a super Chinese rice cultivar with high productivity, good quality and high resistance to both bacterial blight and blast. The embryogenic calli were initiated from mature embroys. It took about 4 months to establish the cell suspenion. The regeneration plants from protoplasts were obtained in 2 months after the isolation of protoplasts.

Cells, Cultured↗

PubMed1990

High frequency plant regeneration from protoplasts of wheat.

Calli were initiated from the mature seeds of wheat (Triticum asetivum L.cv. Xuzhou 211), and suspension cultures were established. The protoplasts isolated from suspension cells were cultured in the modified MS medium solidified with 0.8% agarose. Regenerated cells divided and calli formed. Whole plants were regenerated from protoplast-derived calli. Colony formation was promoted when the medium with lower osmotic pressure was added after two weeks culture. The frequency of regenerated plants was increased with lower concentration of sucrose in the differentiation medium. Shoots were induced effectively with high concentration of cytokinins and calliferous shoots were avoided. The frequency of regenerated plants was affected when protoplast-derived calli were transferred onto the differentiation medium in different periods.

Cells, Cultured↗

PubMed1990

[Protoplast reversion in Bacillus subtilis].

The reversion of the protoplasts of two strains Bac. subtilis on 3 nutritive media: DP of Elliott et al., DM3 of Chang and Cohen and MRG of Bourne and Dancer, is studied. Special attention is devoted to the role of the components gelatin, bovine serum albumin and osmotic stabilizer. In relation to the conclusions drawn in the study a new medium for reversion of protoplasts is suggested--the glucitole reversion agar GRA ensuring continuous 20-40% reversion of the sown protoplasts.

Bacillus subtilis↗

PubMed1990

[Mutagenesis of vitamin B2 producer by using protoplasts].

Studies on preparation, regeneration and ultraviolet-mutagenesis of protoplasts of vitamin B2 producer Eremothecium aohbyii were reported. By using a complex enzyme system (0.5% snial digestase + 0.5% cellulase), a great number of protoplasts were obtained. Ultraviolet-mutagenesising positive mutation ratio is 14.29%. The HPLC analysis indicated that a lot of mutants were screened by using ultraviolet-mutagenesis mutation of protoplasts, Vitamin B2 produced by the mutant E3 increased 116.4%.

Mutation↗