Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.

Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Protoplast Isolation, Culture, and Regeneration in Common and Tartary Buckwheat.

Techniques based on the use of plant protoplasts are a convenient model for better understanding and observing developmental changes in the cells. The establishment of research tools based on protoplasts consists of many steps needed for optimization. Here, we describe the culture of morphogenic callus (MC)- and hypocotyl-derived protoplasts of common (Fagopyrum esculentum Moench) and Tartary (F. tataricum (L.) Gaertn.) buckwheat. Protoplasts embedding in agarose matrix and application of plant hormones, including phytosulfokine (PSK), enable the development of protoplast cultures and plant regeneration.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity.

BACKGROUND: Lignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process. RESULTS: The current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities. CONCLUSION: The results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts.

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

Studying Secretory Protein Synthesis in Nicotiana benthamiana Protoplasts.

Transient gene expression in plant protoplasts facilitates the analysis of hybrid genes in a fast and reproducible manner. The technique is particularly powerful when studying basic conserved biochemical processes including de novo protein synthesis, modification, assembly, transport, and turnover. Unlike individual plants, protoplast suspensions can be divided into almost identical aliquots, allowing the analysis of independent variables with uncertainties restricted to minor pipetting errors/variations. Using the examples of protein secretion and ER retention, we describe the most advanced working practice of routinely preparing, electroporating, and analyzing Nicotiana benthamiana protoplasts. A single batch of electroporation-competent protoplasts permits up to 30 individual transfections. This is ideal to assess the influence of independent variables, such as point mutations, deletions or fusions, or the influence of a co-expressed effector gene in dose-response studies.

Development of PEG-mediated genetic transformation and gene editing system of Bryum argenteum as an abiotic stress tolerance model plant.

KEY MESSAGE: To establish a sterile culture system and protoplast regeneration system for Bryum argenteum, and to establish and apply CRISPR/Cas9 system in Bryum argenteum. Bryum argenteum is a fascinating, cosmopolitan, and versatile moss species that thrives in various disturbed environments. Because of its comprehensive tolerance to the desiccation, high UV and extreme temperatures, it is emerging as a model moss for studying the molecular mechanisms underlying plant responses to abiotic stresses. However, the lack of basic tools such as gene transformation and targeted genome modification has hindered the understanding of the molecular mechanisms underlying the survival of B. argenteum in different environments. Here, we reported the protonema of B. argenteum can survive up to 95.4% water loss. In addition, the genome size of B. argenteum is approximately 313 Mb by kmer analysis, which is smaller than the previously reported 700 Mb. We also developed a simple method for protonema induction and an efficient protoplast isolation and regeneration protocol for B. argenteum. Furthermore, we established a PEG-mediated protoplast transient transfection and stable transformation system for B. argenteum. Two homologues of ABI3(ABA-INSENSITIVE 3) gene were successfully cloned from B. argenteum. To further investigate the function of the ABI3 gene in B. argenteum, we used the CRISPR/Cas9 genetic editing system to target the BaABI3A and BaABI3B gene in B. argenteum protoplasts. This resulted in mutagenesis at the target in about 2-5% of the regenerated plants. The isolated abi3a and abi3b mutants exhibited increased sensitivity to desiccation, suggesting that BaABI3A and BaABI3B play redundant roles in desiccation stress. Overall, our results provide a rapid and simple approach for molecular genetics in B. argenteum. This study contributes to a better understanding of the molecular mechanisms of plant adaptation to extreme environmental.

A high-efficient protoplast transient system for screening gene editing elements in Salvia miltiorrhiza.

KEY MESSAGE: A high-efficiency protoplast transient system was devised to screen genome editing elements in Salvia miltiorrhiza. Medicinal plants with high-value pharmaceutical ingredients have attracted research attention due to their beneficial effects on human health. Cell wall-free protoplasts of plants can be used to evaluate the efficiency of genome editing mutagenesis. The capabilities of gene editing in medicinal plants remain to be fully explored owing to their complex genetic background and shortfall of suitable transformation. Here, we took the Salvia miltiorrhiza as a representative example for developing a method to screen favorable gene editing elements with high editing efficiency in medical plants by a PEG-mediated protoplast transformation. Results indicated that using the endogenous SmU6.1 of S. miltiorrhiza to drive sgRNA and the plant codon-optimized Cas9 driven by the promoter SlEF1α can enhance the efficiency of editing. In summary, we uncover an efficacious transient method for screening editing elements and shed new light on increasing gene editing efficiency in medicinal plants.

Bacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages for enhanced cancer immunotherapy.

The CRISPR-Cas9 system offers substantial potential for cancer therapy by enabling precise manipulation of key genes involved in tumorigenesis and immune response. Despite its promise, the system faces critical challenges, including the preservation of cell viability post-editing and ensuring safe in vivo delivery. To address these issues, this study develops an in vivo CRISPR-Cas9 system targeting tumor-associated macrophages (TAMs). We employ bacterial protoplast-derived nanovesicles (NVs) modified with pH-responsive PEG-conjugated phospholipid derivatives and galactosamine-conjugated phospholipid derivatives tailored for TAM targeting. Utilizing plasmid-transformed E. coli protoplasts as production platforms, we successfully load NVs with two key components: a Cas9-sgRNA ribonucleoprotein targeting Pik3cg, a pivotal molecular switch of macrophage polarization, and bacterial CpG-rich DNA fragments, acting as potent TLR9 ligands. This NV-based, self-assembly approach shows promise for scalable clinical production. Our strategy remodels the tumor microenvironment by stabilizing an M1-like phenotype in TAMs, thus inhibiting tumor growth in female mice. This in vivo CRISPR-Cas9 technology opens avenues for cancer immunotherapy, overcoming challenges related to cell viability and safe, precise in vivo delivery.

Multiplexed effector screening for recognition by endogenous resistance genes using positive defense reporters in wheat protoplasts.

Plant resistance (R) and pathogen avirulence (Avr) gene interactions play a vital role in pathogen resistance. Efficient molecular screening tools for crops lack far behind their model organism counterparts, yet they are essential to rapidly identify agriculturally important molecular interactions that trigger host resistance. Here, we have developed a novel wheat protoplast assay that enables efficient screening of Avr/R interactions at scale. Our assay allows access to the extensive gene pool of phenotypically described R genes because it does not require the overexpression of cloned R genes. It is suitable for multiplexed Avr screening, with interactions tested in pools of up to 50 Avr candidates. We identified Avr/R-induced defense genes to create a promoter-luciferase reporter. Then, we combined this with a dual-color ratiometric reporter system that normalizes read-outs accounting for experimental variability and Avr/R-induced cell death. Moreover, we introduced a self-replicative plasmid reducing the amount of plasmid used in the assay. Our assay increases the throughput of Avr candidate screening, accelerating the study of cellular defense signaling and resistance gene identification in wheat. We anticipate that our assay will significantly accelerate Avr identification for many wheat pathogens, leading to improved genome-guided pathogen surveillance and breeding of disease-resistant crops.

Pseudovampyrella gen. nov.: A genus of Vampyrella-like protoplast extractors finds its place in the Leptophryidae.

Vampyrellid amoebae are predatory protists, which consume a variety of eukaryotic prey and inhabit freshwater, marine and terrestrial ecosystems. Although they have been known for almost 150 years, much of their diversity lacks an in-depth characterization. To date, environmental sequencing data hint at several uncharacterized lineages, to which no phenotype is associated. Furthermore, there are numerous historically described species without any molecular information. This study reports on two new vampyrellid strains from moorlands, which extract the protoplasts of Closterium species (Zygnematophyceae). Our data on morphology, prey range specificity and feeding strategy reveal that the studied vampyrellids are very similar to the historically described Vampyrella closterii. However, phylogenetic analyses demonstrate that the two strains do not belong to the genus Vampyrella and, instead, form a distinct clade in the family Leptophryidae. Hence, we introduce a new genus of algivorous protoplast extractors, Pseudovampyrella gen. nov., with the species P. closterii (= V. closterii) and P. minor. Our findings indicate that the genetic diversity of morphologically described vampyrellid species might be hugely underrated.

Nutritive tissue rich in reserves in the cell wall and protoplast: the case of Manihot esculenta (Euphorbiaceae) galls induced by Iatrophobia brasiliensis (Diptera, Cecidomyiidae).

The galls can offer shelter, protection, and an adequate diet for the gall-inducing organisms. Herein, we evaluated the structure of Manihot esculenta leaves and galls induced by Iatrophobia brasiliensis in order to identify metabolic and cell wall composition changes. We expected to find a complex gall with high primary metabolism in a typical nutritive tissue. Non-galled leaves and galls were subjected to anatomical, histochemical, and immunocytochemical analyses to evaluate the structural features, primary and secondary metabolites, and glycoproteins, pectins, and hemicelluloses in the cell wall. The gall is cylindric, with a uniseriate epidermis, a larval chamber, and a parenchymatic cortex divided into outer and inner compartments. The outer compartment has large cells with intercellular spaces and stocks starch and is designated as storage tissue. Reducing sugars, proteins, phenolic compounds, and alkaloids were detected in the protoplast of inner tissue cells of galls, named nutritive tissue, which presents five layers of compact small cells. Cell walls with esterified homogalacturonans (HGs) occurred in some cells of the galls indicating the continuous biosynthesis of HGs. For both non-galled leaves and galls, galactans and xyloglucans were broadly labeled on the cell walls, indicating a cell growth capacity and cell wall stiffness, respectively. The cell wall of the nutritive tissue had wide labeling for glycoproteins, HGs, heteroxylans, and xyloglucans, which can be used as source for the diet of the galling insect. Manihot esculenta galls have compartments specialized in the protection and feeding of the galling insect, structured by nutritive tissue rich in resource compounds, in the cell walls and protoplast.

Isolation, Purification, and Application of Protoplasts and Transient Expression Systems in Plants.

Protoplasts, derived from plant cells, exhibit remarkable totipotency and hold significant value across a wide spectrum of biological and biotechnological applications. These versatile applications encompass protein subcellular localization and interaction analysis, gene expression regulation, functional characterization, gene editing techniques, and single-cell sequencing. Protoplasts' usability stems from their inherent accessibility and their ability to efficiently incorporate exogenous genes. In this review, we provide a comprehensive overview, including details on isolation procedures and influencing factors, purification and viability assessment methodologies, and the utilization of the protoplast transient expression system. The aim is to provide a comprehensive overview of current applications and offer valuable insights into protoplast isolation and the establishment of transient expression systems in a diverse range of plant species, thereby serving as a valuable resource for the plant science community.

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Preparation of leaf protoplasts from Populus (Populus × xiaohei T. S. Hwang et Liang) and establishment of transient expression system.

Poplar, as a typical woody plant, is an ideal raw material for the production of lignocellulose biofuel. However, the longer life cycle is not conducive to the rapid identification of poplar genes. At present, protoplasts have been used for gene function identification and high-throughput analysis in many model plants. In this paper, a simplified and efficient protoplast isolation and transient expression system of Populus (Populus × xiaohei T. S. Hwang et Liang) is described. Firstly, we proposed an efficient enzyme hydrolysis method for isolating protoplasts from leaves of Populus × xiaohei. Secondly, we optimized the conditions of protoplast transformation mediated by PEG, and established an efficient transient expression system of protoplasts of Populus × xiaohei. Finally, the subcellular localization of three identified Dof transcription factors (PnDof19, PnDof20 and PnDof30) was also observed in the nucleus by using this scheme, which proved that the method was feasible. In general, this efficient method of protoplast isolation and transformation can be used for the study of protein subcellular localization and can be applied to other fields of molecular biology, such as protein interaction, gene activation and so on.

Generation of protoplasts provides a powerful experimental research tool for biological and pathogenicity studies of Pythium insidiosum.

INTRODUCTION: Pythiosis is a high-mortality infectious condition in humans and animals. The etiologic agent is Pythium insidiosum. Patients present with an ocular, vascular, cutaneous/subcutaneous, or gastrointestinal infection. Antifungal medication often fails to fight against P. insidiosum. The effective treatment is limited to radical surgery, resulting in organ loss. Fatal outcomes are observed in advanced cases. Pythiosis needs to be studied to discover novel methods for disease control. Genome data of P. insidiosum is publicly available. However, information on P. insidiosum biology and pathogenicity is still limited due to the lack of a cost-effective animal model and molecular tools. MATERIALS AND METHODS: We aimed to develop a high-efficiency protocol for generating P. insidiosum protoplast, and used it to set up an animal model, in vitro drug susceptibility assay, and DNA transformation for this pathogen. RESULTS: P. insidiosum protoplast was successfully generated to establish a feasible pythiosis model in embryonic chicken eggs and an efficient in vitro drug susceptibility assay. DNA transformation is a critical method for gene manipulation necessary for functional genetic studies in pathogens. Attempts to establish a DNA transformation method for P. insidiosum using protoplast were partly successful. Significant work needs to be done for genetically engineering a more robust selection marker to generate stable transformants at increased efficiency. CONCLUSION: This study is the first to report an efficient P. insidiosum protoplast production for clinical and research applications. Such advances are crucial to speeding up the pathogen's biology and pathogenicity exploration.

Single Cell RNA-Sequencing in Arabidopsis Root Tissues.

Droplet-based single-cell RNA-sequencing (scRNA-seq) empowers transcriptomic profiling with an unprecedented resolution, facilitating insights into the cellular heterogeneity of tissues, developmental progressions, stress-response dynamics, and more at single-cell level. In this chapter, we describe the experimental workflow of processing Arabidopsis root tissue into protoplasts and generating single-cell transcriptomes. We also describe the general computational workflow of visualizing and utilizing scRNA-seq data. This protocol can be used as a starting point for establishing a scRNA-seq workflow.

CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

Integrated flow cytometric and proteomics analyses reveal the regulatory network underlying sugarcane protoplast responses to fusion.

BACKGROUND: Somatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and molecular mechanisms that shape protoplast responses to fusion. METHOD: We employed flow cytometry, cytology, proteomics, and gene expression analysis to examine the sugarcane (Saccharum spp.) protoplast fusion. RESULTS: Flow cytometry analysis revealed the fusion rate of protoplasts was 1.95%, the FSC value and SSC of heterozygous cells was 1.17-1.47 times higher than that of protoplasts. The protoplasts viability decreased and the MDA increased after fusion. During fusion, the cell membranes were perforated to different degrees, nuclear activity was weakened, while microtubules depolymerized and formed several short rod like structures in the protoplasts. The most abundant proteins during fusion were mainly involved in RNA processing and modification, cell cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Moreover, the expression of key regeneration genes, such as WUS, GAUT, CESA, PSK, Aux/IAA, Cdc2, Cyclin D3, Cyclin A, and Cyclin B, was significantly altered following fusion. PURPOSE AND SIGNIFICANCE: Overall, our findings provide a theoretical basis that increases our knowledge of the mechanisms underlying protoplast fusion.