PEG-mediated Protoplast Transformation Research Articles

Advancing Cordyceps militaris Industry: Gene Manipulation and Sustainable Biotechnological Strategies.

Cordyceps militaris is considered to be of great medicinal potential due to its remarkable pharmacological effects, safety, and edible characteristics. With the completion of the genome sequence and the advancement of efficient gene-editing technologies, coupled with the identification of gene functions in Cordyceps militaris, this fungus is poised to emerge as an outstanding strain for medicinal engineering applications. This review focuses on the development and application of genomic editing techniques, including Agrobacterium tumefaciens-mediated transformation (ATMT), PEG-mediated protoplast transformation (PMT), and CRISPR/Cas9. Through the application of these techniques, researchers can engineer the biosynthetic pathways of valuable secondary metabolites to boost yields; such metabolites include cordycepin, polysaccharides, and ergothioneine. Furthermore, by identifying and modifying genes that influence the growth, disease resistance, and tolerance to environmental stress in Cordyceps militaris, it is possible to stimulate growth, enhance desirable traits, and increase resilience to unfavorable conditions. Finally, the green sustainable industrial development of C. militaris using agricultural waste to produce high-value-added products and the future research directions of C. militaris were discussed. This review will provide future directions for the large-scale production of bioactive ingredients, molecular breeding, and sustainable development of C. militaris.

Phytosulfokine alpha enhances regeneration of transformed and untransformed protoplasts of Brassica oleracea.

Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of in vivo plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on in vitro cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four Brassica oleracea cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.

Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing

BackgroundSqualene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids.ResultIn this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach.ConclusionThe findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.

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

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.

CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species.

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of Sporothrix species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in S. brasiliensis, S. schenckii, and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene pks1, which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU80 homolog that is critical for non-homologous end-joining DNA repair. The use of Δku80 strains from S. brasiliensis enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species. IMPORTANCE Sporotrichosis caused by Sporothrix brasiliensis is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in Sporothrix, this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of Sporothrix species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.

Establishment of protoplasts transient expression system in Pinellia ternata (Thunb.) Breit.

In this study, we established an efficient and rapid transient expression system in the protoplasts of Pinellia ternata (Thunb.) Breit. (P. ternata). The protoplasts of P. ternata were prepared from plant leaves as the source material by digesting them with the combination of 20g·l-1 cellulase and 15g·l-1 macerozyme for 6h. Based on the screening of PEG concentration, the conditions for PEG-mediated protoplast transformation were improved, and the highest transformation efficiency was found for 40% PEG 4000. Furthermore, we used the subcellular protein localization technique in P. ternata protoplasts to allow further validation of transient expression system. We present the method that can be applicable for studying both gene verification and expression in P. ternata protoplasts, thus allowing for engineering the improved varieties of P. ternata through molecular plant breeding techniques. This method can also be widely applicable for analyzing protein interactions, detecting promoter activity, for somatic cell fusion in plant breeding, as well as for other related studies.

Efficient PEG-mediated transformation of oil palm mesophyll protoplasts and its application in functional analysis of oil palm promoters

Efficient PEG-mediated transformation of oil palm mesophyll protoplasts and its application in functional analysis of oil palm promoters

Pectate Lyase Genes Abundantly Expressed During the Infection Regulate Morphological Development of Colletotrichum camelliae and CcPEL16 Is Required for Full Virulence to Tea Plants.

Colletotrichum camelliae is the dominant species causing foliar diseases of tea plants (Camellia sinensis) in China. Transcriptome data and reverse transcription-quantitative PCR (qRT-PCR) analysis have demonstrated that the pectate lyase genes in C. camelliae (CcPELs) were significantly upregulated during infectious development on tea plants (cv. Longjing43). To further evaluate the biological functions of CcPELs, we established a polyethylene glycol (PEG)-mediated protoplast transformation system of C. camelliae and generated targeted deletion mutants of seven CcPELs. Phenotypic assays showed that the genes contribute to mycelial growth, conidiation, and appressorium development. The polypeptides encoded by each CcPEL gene contained a predicted N-terminal signal peptide, and a yeast invertase secretion assay suggested that each CcPEL protein could be secreted. Cell death-suppressive activity assays confirmed that all seven CcPELs did not suppress Bax-induced cell death in tobacco leaf cells. However, deletion of CcPEL16 significantly reduced necrotic lesions on tea leaves. Taken together, these results indicated that CcPELs play essential roles in regulating morphological development, and CcPEL16 is required for full virulence in C. camelliae. IMPORTANCE In this study, we first established a PEG-mediated protoplast transformation system of C. camelliae and used it to investigate the biological functions of seven pectate lyase genes (CcPELs) which were abundantly expressed during infection. The results provided insights into the contributions of pectate lyase to mycelial growth, conidial production, appressorium formation, and the pathogenicity of C. camelliae. We also confirmed the secretory function of CcPEL proteins and their role in suppressing Bax-induced cell death. Overall, this study provides an effective method for generating gene-deletion transformants in C. camelliae and broadens our understanding of pectate lyase in regulating morphological development and pathogenicity.

Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.

Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1nt deletion (6 mutants), 3 nt deletion with substitution of 1nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.

Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes

BackgroundCRISPR/Cas9 is the most powerful and versatile genome-editing tool that permits multiplexed-targeted gene modifications for the genetic enhancement of oil palm. Multiplex genome-editing has recently been developed for modifying multiple loci in a gene or multiple genes in a genome with high precision. This study focuses on the development of high-oleic oil palm, the primary target trait for healthy low-saturated oil. To achieve this, the fatty acid desaturase 2 (FAD2) and palmitoyl-acyl carrier protein thioesterase (PAT) genes, both of which are associated with fatty acid metabolism biosynthesis pathways in oil palm, need to be knocked out. The knockout of FAD2 and PAT leads to an accumulation of oleic acid content in oil palms. ResultsA total of four single-guide RNAs (sgRNAs) were designed in silico based on the genomic sequences of EgFAD2 and EgPAT. Using robust plant CRISPR/Cas9 vector technology, multiple sgRNA expression cassettes were efficiently constructed into a single-binary CRISPR/Cas9 vector to edit the EgFAD2 and EgPAT genes. Each of the constructed transformation vectors was then delivered into oil palm embryogenic calli using the biolistic, Agrobacterium-mediated, and PEG-mediated protoplast transformation methods. Sequence analysis of PCR products from 15 samples confirmed that mutations were introduced at four target sites of the oil palm EgFAD2 and EgPAT genes. Single- and double-knockout mutants of both genes were generated, with large and small deletions within the targeted regions. Mutations found at EgFAD2 and EgPAT target sites indicate that the Cas9/sgRNA genome-editing system effectively knocked out both genes in oil palm. ConclusionThis technology is the first in oil palm to use CRISPR/Cas9 genome-editing to target high-oleic-associated genes. These findings showed that multiplex genome-editing in oil palm could be achieved using multiple sgRNAs. Targeted mutations detected establish that the CRISPR/Cas9 technology offers a great potential for oil palm.

Gene Replacement by a Selectable Marker in the Filamentous FungusMagnaporthe oryzae.

Magnaporthe oryzaeis a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain. Key features Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.

Evolution of Benzimidazole Resistance Caused by Multiple Double Mutations of β-Tubulin in Corynespora cassiicola.

Cucumber target leaf spot caused by Corynespora cassiicola has devastated greenhouse cucumber production. In our previous study, the resistance monitoring of C. cassiicola to carbendazim was carried out, and a large number of resistant populations carrying various mutations (M163I&E198A, F167Y&E198A, F200S&E198A, or E198A) in β-tubulin were detected. However, the single-point mutations M163I, F167Y, and F200S have remained undetected. To investigate the evolutionary mechanism of double mutations in β-tubulin of C. cassiicola resistance to benzimidazoles, site-directed mutagenesis was used to construct alleles with corresponding mutation genotypes in β-tubulin. Through PEG-mediated protoplast transformation, all the mutants except for the M163I mutation were obtained and conferred resistance to benzimidazoles. It was found that the mutants conferring the E198A or double-point mutations showed high resistance to carbendazim and benomyl, but the mutants conferring the F167Y or F200S mutations showed moderate resistance. Except, the F200S mutants showed low resistance, the resistance level of the other mutants to thiabendazole seemed no difference. In addition, compared to the other mutants, the F167Y and F200S mutants suffered a more severe fitness penalty in mycelial growth, sporulation, and virulence. Thus, combined with the resistance level, fitness, and molecular docking results, we concluded that the field double mutations (F167Y&E198A and F200S&E198A) evolved from the single mutations F167Y and F200S, respectively.

Establishment and optimization of PEG-mediated protoplast transformation in the microalga Haematococcus pluvialis

Genetic manipulation of Haematococcus pluvialis is difficult because of the lack of a stable and convenient transformation system. The pH124-EGFP-Ble vector containing ble as a selective gene and EGFP as a reporter gene was constructed and employed for effective transformation. H. pluvialis protoplasts were obtained by treating with cellulase and macerozeme. Then polyethylene glycol-mediated transformation was established by incubating the protoplast with the vector. To improve the transformation efficiency of H. pluvialis protoplasts, the transformation system was optimized in consideration of different influencing factors, including zeomycin concentration, growth stage, amount of transformed vector, linearization of the vector, and duration of low-intensity illumination. The integration and expression of ble and EGFP was confirmed in the transformants. Moreover, the optimal combination for protoplast transformation of H. pluvialis was determined to be 5 µg of the linearized vector used to transform cells in the log growth phase, and then the transformed protoplasts allowed to recover under low-intensity illumination for 6 h. This study represents and describes the successful development of an H. pluvialis transformation protocol using protoplasts, which will enable convenient genetic manipulation of this important algal species.

Development of CRISPR-Cas9 genome editing system in Talaromyces marneffei

Talaromyces marneffei is an important pathogenic thermally dimorphic fungus causing systemic talaromycosis mainly prevalent in Southeast Asia. The dimorphic transition between mycelium and yeast is considered crucial for the pathogenicity of T. marneffei. However, the lack of genetic toolbox has been a major impediment for understanding its pathogenicity. Here a CRISPR-Cas9 system was developed to facilitate genetic manipulations in this organism. In this study, the CRISPR-Cas9 gene editing system uses a native U6 snRNA promoter from T. marneffei to drive the expression of sgRNA. Employing this system and PEG-mediated protoplast transformation, the sakA gene was mutated. Sanger sequencing confirmed nearly 40% site-directed mutation rate. The phenotype analysis confirmed the sakA gene function in T. marneffei dimorphic transition. Our study provided a powerful genome-manipulating tool, which could accelerate studies on T. marneffei for further revealing the mechanisms of its pathogenicity.

Plastid Transformation in Physcomitrium (Physcomitrella) patens: An Update.

The moss Physcomitrium (Physcomitrella) patens performs efficient homologous recombination in both the nucleus and plastid enabling the study of individual gene function by generating precise inactivation or modification of genes. Polyethylene glycol (PEG)-mediated transformation of protoplasts is routinely used to study the nuclear gene function of P. patens. PEG-mediated protoplast transformation is also applied for plastid transformation of this moss. The efficiency of plastid transformation is quite reliable and one or two homoplasmic transplastomic lines are obtained in a plastid transformation experiment (5×105 protoplasts) by selection for spectinomycin resistance.

CRISPR/Cas9-Mediated Genome Editing of Trichoderma reesei.

In this protocol, we describe the establishment of a CRISPR/Cas9 system in Trichoderma reesei by generating a specific, codon-optimized Cas9-expressing strain and by in vitro transcription of a gRNA. This system induces mutagenesis or introduces a gene in a targeted way based on PEG-mediated protoplast transformation. Up to three targets, multiplexed genome editing can be obtained in one transformation.

Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana

BackgroundTo date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system.ResultsProtoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems.ConclusionsThe PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.

Isolation of leaf mesophyll protoplasts optimized by orthogonal design for transient gene expression in Catalpa bungei

Catalpa bungei is an important ornamental plant and timber species that originated in China. With the forthcoming C. bungei genome sequence, an efficient and convenient transformation system should be developed. The transient gene expression system using protoplasts is fast and efficient and can be applied to study the functions of genes in C. bungei. In this study, the main factors influencing the isolation of protoplasts from C. bungei were optimized by orthogonal design. The results showed that when the enzymolysis solution contained 3% Cellulase RS (w/v), 2% Macerozyme R-10 (w/v), and 0.4 M mannitol and the enzymolysis time was 8 h, the protoplast yield was 1 × 106 protoplasts/g fresh weight [FW]. The viability of protoplasts was approximately 90 % under optimal enzymolysis conditions. Furthermore, using GFP as the reporter gene, we verified the feasibility of PEG-mediated protoplast transformation of C. bungei. We established an efficient protoplast isolation procedure and feasible transient gene expression system. This methodology, when combined with genetics and omics techniques, will allow further study of the functions of the genes in C. bungei.

Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations.

We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus.

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae.

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.