Protoplasts of two different auxotrophic mutants of Streptomyces lavendulae were fused with polyethylene glycol (PEG) 1,000, and allowed to regenerate on selective media. Prototrophic colonies overwhelmed other types of recombinants on any selective media. These prototrophic strains were stable after successive isolation. These results suggest that stable diploid cells were formed by cell fusion, which differed from the case of S. coelicolor.

No abstract available

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

The phytohormone jasmonic acid (JA) plays an important role in various plant developmental processes and environmental adaptations. The JA signaling pathway has been well-elucidated in the reference plant Arabidopsis thaliana. It starts with the perception of the active JA derivative, jasmonoyl-isoleucine (JA-Ile), by the F-box protein COI1 which is part of the E3-ligase SCFCOI1. Binding of JA-Ile enables the interaction between COI1 and JAZ repressor proteins. Subsequent degradation of JAZ proteins leads to the activation of transcription factors like e.g., MYC2. Here we demonstrate that the pathway can be reconstituted in transiently transformed protoplasts. Analysis of the stability of a JAZ1-fLuc fusion protein as a function of COI1 transiently expressed in coi1 protoplasts allows structure function analysis of both JAZs and COI1. Using this system, we found that conserved cysteines in COI1 influence steady state COI1 protein levels. Using a luciferase reporter gene under the control of the JAZ1 promoter enable to address those features of JAZ1 that are required for MYC2 repression. Interestingly, the conserved TIFY-motif previously described to interact with NINJA to recruit the corepressor TOPLESS is not necessary for repression. This result is in favor of the alternative repression mode that proposes a direct competition between repressive JAZs and promotive MEDIATOR25 at MYC2. Finally, using protoplasts from the aoscoi1 double mutant, which is deficient in JA synthesis and perception, we provide a system that has the potential to study the activity of different COI1 variants in the presence of different ligands.

The complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays.

Differentiated plant cells can retain the capacity to be reprogrammed into pluripotent stem cells during regeneration. This capacity is associated with both cell cycle reactivation and acquisition of specific cellular characters. However, the molecular mechanisms underlying the reprogramming of protoplasts into stem cells remain largely unknown. Protoplasts of the moss Physcomitrella patens easily regenerate into protonema and therefore provide an ideal system to explore how differentiated cells can be reprogrammed to produce stem cells.

The spheroplasts and protoplasts of cell wall-deficient (CWD) bacteria are able to revert to their original cellular morphologies through the regeneration of their cell walls. However, whether this is true for giant protoplasts (GPs), which can be as large as 10 μm in diameter, is unknown. GPs can be prepared from various bacteria, including Escherichia coli and Bacillus subtilis, and also from fungi, through culture in the presence of inhibitors for cell wall synthesis or mitosis. In this report, we prepared GPs from E. coli and showed that they can return to rod-shaped bacterium, and that they are capable of colony formation. Microscopic investigation revealed that the regeneration process took place through a variety of morphological pathways. We also report the relationship between GP division and GP volume. Finally, we show that FtsZ is crucial for GP division. These results indicate that E. coli is a highly robust organism that can regenerate its original form from an irregular state, such as GP.

We demonstrate that plasma membrane biosynthesis and vacuole formation require DNA replication in Enterococcus faecalis protoplasts. The replication inhibitor novobiocin inhibited not only DNA replication but also cell enlargement (plasma membrane biosynthesis) and vacuole formation during the enlargement of the E. faecalis protoplasts. After novobiocin treatment prior to vacuole formation, the cell size of E. faecalis protoplasts was limited to 6 μm in diameter and the cells lacked vacuoles. When novobiocin was added after vacuole formation, E. faecalis protoplasts grew with vacuole enlargement; after novobiocin removal, protoplasts were enlarged again. Although cell size distribution of the protoplasts was similar following the 24 h and 48 h novobiocin treatments, after 72 h of novobiocin treatment there was a greater number of smaller sized protoplasts, suggesting that extended novobiocin treatment may inhibit the re-enlargement of E. faecalis protoplasts after novobiocin removal. Our findings demonstrate that novobiocin can control the enlargement of E. faecalis protoplasts due to inhibition of DNA replication.

Protoplasts of Enterococcus faecalis did not divide but enlarged in Difco Marine Broth containing penicillin. Our previous studies have demonstrated that transcription and translation were essential for bacterial cell enlargement. However, it was uncertain whether replication was also essential. In this study, we measured the amount of DNA in E. faecalis cells during the course of enlargement using quantitative polymerase chain reaction. The growth of normally divided cells (native forms) of E. faecalis exhibited a log phase before 6 h of incubation was reached. Although a difference in quantitation cycle (Cq) values between the replication initiation and termination regions was observed in the log phase, it was not present in the stationary growth phase. On the other hand, the amount of DNA in E. faecalis protoplasts increased during the cell enlargement incubation. The difference of Cq values between the protoplasts at 0 and 96 h of incubation was 8-9, indicating that the DNA amount at 96 h was 200-500 times higher than that at 0 h. The Cq values differed between the replication initiation and termination regions, indicating that the replication level was high. When novobiocin, a DNA replication inhibitor, was added to the medium at 24 h of incubation, DNA replication and cell enlargement were almost stopped. Thus, replication plays an important role in the enlargement of E. faecalis protoplasts.

Extracellular vesicles (EVs) are membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties in perceiving the role of EVs during the fungal life, and particularly in cell wall biogenesis, is caused by the presence of a thick cell wall. One alternative to have better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid/sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that (i) EV production is a common feature of different morphological stages of this major fungal pathogen and (ii) protoplastic EVs are promising tools for undertaking studies of vesicle functions in fungal cells.IMPORTANCE Fungal cells use extracellular vesicles (EVs) to export biologically active molecules to the extracellular space. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate the role of EV production in cell wall biogenesis. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our report is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts represent a promising model for functional studies of fungal vesicles.

During heterologous protein production using recombinant microbes, the protein tends to accumulate in the cell and may not be secreted. Here, we studied the production of secretory cutinase (heterologous protein) by recombinant Saccharomyces cerevisiae protoplasts.

Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato (Solanum lycopersicum) cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from third to fourth true leaves and cultured at an optimal density of 1 × 105 protoplasts/ml. We determined the optimal culture media for protoplast proliferation, callus formation, de novo shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications, such as genome engineering, as well as basic research on plant regeneration in Solanaceae species.

Proximity-dependent biotin identification (BioID), which detects physiologically relevant proteins based on the proximity-dependent biotinylation process, has been successfully used in different organisms. In this report, we established the BioID system in rice protoplasts. Biotin ligase BirAG was obtained by removing a cryptic intron site in the BirA∗ gene when expressed in rice protoplasts. We found that protein biotinylation in rice protoplasts increased with increased expression levels of BirAG. The biotinylation effects can also be achieved by exogenous supplementation of high concentrations of biotin and long incubation time with protoplasts. By using this system, multiple proteins were identified that associated with and/or were proximate to OsFD2 in vivo. Our results suggest that BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment in plant cell.

The ability to induce Arabidopsis protoplasts to dedifferentiate and divide provides a convenient system to analyze organelle dynamics in plant cells acquiring totipotency. Using peroxisome-targeted fluorescent proteins, we show that during protoplast culture, peroxisomes undergo massive proliferation and disperse uniformly around the cell before cell division. Peroxisome dispersion is influenced by the cytoskeleton, ensuring unbiased segregation during cell division. Considering their role in oxidative metabolism, we also investigated how peroxisomes influence homeostasis of reactive oxygen species (ROS). Protoplast isolation induces an oxidative burst, with mitochondria the likely major ROS producers. Subsequently ROS levels in protoplast cultures decline, correlating with the increase in peroxisomes, suggesting that peroxisome proliferation may also aid restoration of ROS homeostasis. Transcriptional profiling showed up-regulation of several peroxisome-localized antioxidant enzymes, most notably catalase (CAT). Analysis of antioxidant levels, CAT activity and CAT isoform 3 mutants (cat3) indicate that peroxisome-localized CAT plays a major role in restoring ROS homeostasis. Furthermore, protoplast cultures of pex11a, a peroxisome division mutant, and cat3 mutants show reduced induction of cell division. Taken together, the data indicate that peroxisome proliferation and CAT contribute to ROS homeostasis and subsequent protoplast division induction.

Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology.

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors-temperature, enzyme lysis time, and concentration of the lysing enzyme-were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 10⁷ protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46⁻54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

Genome-editing is being implemented in increasing number of plant species using engineered sequence specific nucleases (SSNs) such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9), Transcription activator like effector nucleases (TALENs), and more recently CRISPR/Cas12a. As the tissue culture and regeneration procedures to generate gene-edited events are time consuming, large-scale screening methodologies that rapidly facilitate validation of genome-editing reagents are critical. Plant protoplast cells provide a rapid platform to validate genome-editing reagents. Protoplast transfection with plasmids expressing genome-editing reagents represents an efficient and cost-effective method to screen for in vivo activity of genome-editing constructs and resulting targeted mutagenesis. In this study, we compared three existing methods for detection of editing activity, the T7 endonuclease I assay (T7EI), PCR/restriction enzyme (PCR/RE) digestion, and amplicon-sequencing, with an alternative method which involves tagging a double-stranded oligodeoxynucleotide (dsODN) into the SSN-induced double stranded break and detection of on-target activity of gene-editing reagents by PCR and agarose gel electrophoresis. To validate these methods, multiple reagents including TALENs, CRISPR/Cas9 and Cas9 variants, eCas9(1.1) (enhanced specificity) and Cas9-HF1 (high-fidelity1) were engineered for targeted mutagenesis of Acetolactate synthase1 (ALS1), 5-Enolpyruvylshikimate- 3-phosphate synthase1 (EPSPS1) and their paralogs in potato. While all methods detected editing activity, the PCR detection of dsODN integration provided the most straightforward and easiest method to assess on-target activity of the SSN as well as a method for initial qualitative evaluation of the functionality of genome-editing constructs. Quantitative data on mutagenesis frequencies obtained by amplicon-sequencing of ALS1 revealed that the mutagenesis frequency of CRISPR/Cas9 reagents is better than TALENs. Context-based choice of method for evaluation of gene-editing reagents in protoplast systems, along with advantages and limitations associated with each method, are discussed.

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by 'Candidatus Phytoplasma mali' (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called "Protein in Malus Expressed 2" (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.

Wounding is a constant threat to plant survival throughout their lifespan; therefore, understanding the biological responses to wounds at the cellular level is important. The protoplast system is versatile for molecular biology, however, no wounding studies on this system have been reported. We established a new approach for wounding research using mechanically damaged Arabidopsis mesophyll protoplasts. Wounded protoplasts showed typical wounding responses, such as increased MPK6 kinase activity and upregulated JAZ1 expression. We also assessed expression profiles and protein stability of the basic helix-loop-helix transcription factor MYC2 in wounded protoplasts. Promoter activity, gene expression, and protein stability of MYC2 were compromised, but recovered in the early stage of wounding. In the late stage, the promoter activity and expression of MYC2 were increased, but the protein stability was not changed. According to the results of the present study, this new cell-based approach will be of use in various molecular studies on plant wounding.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.