Protoplast Reversion Research Articles

Biocontrol with Myxococcus sp. KYC 1126 Against Anthracnose in Hot Pepper

Antifungal activity of three Myxococcus spp., KYC 1126, 1136, and 2001, was tested in vitro against three phytopathogenic fungi (Botrytis cinerea, Colletotrichum acutatum, and Pyricularia grisea). Spore germination and mycelial growth of the three pathogenic fungi were completely inhibited by bioactive substances from a myxobacterium KYC 1126. In addition, the activity of KYC 1126 was fungicidal, but liquid culture filtrate of KYC 1126 did not affect protoplast reversion in C. acutatum. A bioassay of KYC 1126 filtrate against anthracnose in hot pepper was conducted in the greenhouse and field at 2009 and 2010. The incidence of anthracnose in control seedlings was 74%, but was reduced to 29% after KYC 1126 treatment. The control value with KYC 1126 was 60% while that with the fungicide dithianon was 42%. In the greenhouse, disease incidence with KYC 1126 was consistentely 10-35% lower than with fungicide as a positive control. The control value with KYC 1126 was 13.4% and 41.0%, whereas that with the fungicide was 52.3% and 63% in 2009 and 2010, respectively. Although anti-anthracnose activity of KYC 1126 was not maintained for long time in the field, the bacteriolytic myxobacterium KYC 1126 could be a prospective biocontrol agent.

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes

Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10(7)-10(9) protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments.

Cell wall structure and protoplast reversion in basidiomycetes

Cell wall structure and protoplast reversion in basidiomycetes

The influence of congo red on the cell wall and (1----3)-beta-D-glucan microfibril biogenesis in Saccharomyces cerevisiae.

Congo red was applied to growing yeast cells and regenerating protoplasts in order to study its effects on wall biogenesis and cell morphogenesis. In the presence of the dye, the whole yeast cells grew and divided to form chains of connected cells showing aberrant wall structures on both sides of the septum. The wall-less protoplasts in solid medium with the dye exhibited an abnormal increase in volume, regeneration of aberrant cell walls and inability to carry out cytokinesis or protoplast reversion to cells. In liquid medium, the protoplasts synthesized glucan nets composed mainly of thin fibrils orientated at random, whereas normally, in the absence of dye, the nets consist of rather thick fibrils, 10 to 20 nm in width, assembled into broad ribbons. These fibrils are known to consist of triple 6/1 helical strands of (1----3)-beta-D-glucan aggregated laterally in crystalline packing. The thin fibrils (c. 4 to 8 nm wide) can contain only a few triple helical strands (c. 1.6 nm wide) and are supposed to be prevented from further aggregation and crystallization by complexing with Congo red on their surfaces. Some loose triple 6/1 helical strands (native elementary fibrils) are also discernible. They represent the first native (1----3)-beta-D-glucan elementary fibrils depicted by electron microscopy. The effects of Congo red on growth and the wall structure in normal cells and regenerating protoplasts in solid medium can be explained by the presence of a complex which the dye forms with (helical) chain parts of the glucan network and which results in a loss of rigidity by a blocked lateral interaction between the helices.

バチルス細胞融合の平板培養の簡易化 混釈法によるプロトプラスト再生と融合体検出

バチルス細胞融合の平板培養の簡易化 混釈法によるプロトプラスト再生と融合体検出

Autoclaved Polyethylene Glycol decreases yeast protoplast reversion and hybrid production

Autoclaved solutions of the fusogen Polyethylene Glycol (PEG) give reduced yeast protoplast reversion and hybrid formation. Autoclaving has been shown to cause PEG degradation and increase the acetaldehyde content. Consequently, membrane filtration is recommended as the optimum means of sterilising PEG solutions to ensure maximum protoplast reversion and hybrid yield.

Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe.

To clarify the involvement of actin in the formation of the yeast cell wall, reverting protoplasts of Schizosaccharomyces pombe were used as a simple model system. Actin of reverting protoplasts was labeled with rhodamine-conjugated phalloidin and observed by conventional fluorescence microscopy and laser scanning confocal microscopy. A close spatial as well as temporal relationship between actin and cell wall formation was observed in protoplast reversion. That is, the site of actin 'dots' in the reverting protoplasts coincided with the site of new wall formation and the timing of rearrangement of actin coincided with the initiation of cell wall formation and with the timing of cell wall expansion. Treatment of reverting protoplasts with cytochalasin D (CD) further clarified the close relationship between actin and cell wall organization. The effect of CD was dose dependent. A high dose of CD caused the absence of actin as well as the complete inhibition of cell wall formation. A low dose of CD caused weakly stained unlocalized actin, which induced grossly aberrant cell wall deposition as well as substantial changes in the morphology of the reverting protoplasts. These results demonstrated that actin is associated with initiation of cell wall formation, the proper deposition of cell wall materials, and maintaining the normal morphology of reverting protoplasts. Scanning electron microscopy revealed the presence of a fibrillar net structure on the surface of non-treated control reverting protoplasts. However, the absence of a fibrillar network on the surface of reverting protoplasts was observed in the presence of a high concentration of CD. Lack of localization of microfibrils as well as poor development of the fibrillar network were also observed in the presence of a low concentration of CD. Recovery experiments confirmed the close relationship between actin and cell wall formation.

Pleomorphism in beetle-gallery conidial fungi and protoplast reversion.

Pleomorphism in beetle-gallery conidial fungi and protoplast reversion.

Isolation and reversion of protoplasts ofPleurotus sajor-caju

The conditions for protoplast isolation and reversion inPleuro tus sajor-caju were determined. Spherical, osmotically sensitive protoplasts were released from the mycelia through the action of Novozym 234 and cellulase CP in a suitable osmotic solution. A maximum protoplast yield was obtained from young mycelia at an early exponential growth phase by the use of 0.6 mol/l MgSC4 in a 0.01 mol/l phosphate buffer at pH 5.0. Mannitol appeared to be more suitable forPleurotus protoplast reversion. Two patterns of morphological development of the protoplasts were observed in the osmotic liquid medium. The reversion frequency was, however, low at 4–5%. There appeared to be no significant difference in the reversion frequencies of the protoplasts grown on the complete medium and on the minimal medium.

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state

When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by beta-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.

Experimental inhibition of cell wall formation and of reversion in Nadsonia elongata and Schizosaccharomyces pombe protoplasts.

The growth, cell wall regeneration, and the reversion of the protoplasts ofNadsonia elongata andSchizosaccbaromyces pombe cultivated in nutrient media containing snail enzyme was studied by light and electron microscopy. The protoplasts grew in the presence of snail enzyme and an incomplete cell wall composed of fibrils was formed on their surface. Thus, the presence of snail enzyme inhibited the completion of cell wall structure and, consequently, the reversion of the protoplasts to normal cells. The transfer of these protoplasts to medium free from snail enzyme led first to the completion of the cell wall and then to the reversion of the protoplasts to normal cells. The reported experiments confirmed that the regeneration of the complete cell wall preceded the protoplast reversion.