RESUMEN
Parthenocarpy, characterized by seedless fruit development without pollination or fertilization, offers the advantage of consistent fruit formation, even under challenging conditions such as high temperatures. It can be induced by regulating auxin homeostasis; PAD1 (PARENTAL ADVICE-1) is an inducer of parthenocarpy in Solanaceae plants. However, precise editing of PAD1 is not well studied in peppers. Here, we report a highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) for CaPAD1 editing in three valuable cultivars of pepper (Capsicum annuum L.): Dempsey, a gene-editable bell pepper; C15, a transformable commercial inbred line; and Younggo 4, a Korean landrace. To achieve the seedless pepper trait under high temperatures caused by unstable climate change, we designed five single guide RNAs (sgRNAs) targeting the CaPAD1 gene. We evaluated the in vitro on-target activity of the RNP complexes in three cultivars. Subsequently, we introduced five CRISPR/Cas9-RNP complexes into protoplasts isolated from three pepper leaves and compared indel frequencies and patterns through targeted deep sequencing analyses. We selected two sgRNAs, sgRNA2 and sgRNA5, which had high in vivo target efficiencies for the CaPAD1 gene across the three cultivars and were validated as potential off-targets in their genomes. These findings are expected to be valuable tools for developing new seedless pepper cultivars through precise molecular breeding of recalcitrant crops in response to climate change.
Asunto(s)
Sistemas CRISPR-Cas , Capsicum , Edición Génica , Protoplastos , Ribonucleoproteínas , Capsicum/genética , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Protoplastos/metabolismo , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Protoplasts are plant cells from which the pectocellulosic cell wall has been removed, thus keeping the plasma membrane intact. For plant secondary metabolites research, this system is a powerful tool to study the metabolites' dynamics inside the cells, such as the subcellular localization of proteins, characterization of gene function, transcription factors involved in metabolite pathways, protein transport machinery, and to perform single-cell omics studies. Due to its lack of a cell wall, better images of the interior of the cell can be obtained compared to the whole tissue. This allows the identification of specific cell types involved in the accumulation of specialized metabolites, such as alkaloids, given their autofluorescence properties. Here is a simplified protocol to obtain protoplasts from leaves and in vitro cell cultures from Argemone mexicana, which produces the pharmacologically important alkaloids berberine and sanguinarine.
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Alcaloides , Argemone , Plantas Medicinales , Protoplastos , Protoplastos/metabolismo , Argemone/química , Argemone/metabolismo , Plantas Medicinales/metabolismo , Plantas Medicinales/química , Alcaloides/metabolismo , Hojas de la Planta/metabolismo , Benzofenantridinas/metabolismo , Berberina/metabolismo , IsoquinolinasRESUMEN
The effect mechanism of Mn on Cd uptake by Celosia argentea was investigated via a series of hydroponics experiments. The results showed that different manganese treatments had different effects on Cd uptake by C. argentea. Mn pretreatment increased Cd uptake by root protoplasts at Cd concentrations (4 and 6 µM). Protoplasts reached peak Cd uptake rate at 6 µM Cd and 25 °C, with 67.71 ± 0.13 µM h-1 mL-1 in the control, and 77.99 ± 0.49 µM h-1 mL-1 in the 50 µM Mn pretreatment group. However, simultaneous treatment with Cd and Mn reduced the Cd2+ uptake by root protoplasts. This discrepancy may be attributed to the fact that cadmium and manganese share some transporters in root cells. The transcriptome analysis in roots revealed that ten genes (including ABCC, ABCA, ABCG, ABCB, ABC1, BZIP19, and ZIP5) were significantly upregulated in response to Mn stress (p < 0.05). These genes regulate the expression of transporters belonging to the ABC, and ZIP families, which may be involved in Cd uptake by root cells of C. argentea. Mn pretreatment upregulates the expression of Mn/Cd transporters, enhancing Cd uptake by root protoplasts. For the simultaneous treatment of Cd and Mn, inhibition of Cd uptake was due to the competition of the same transporters. These findings provide helpful insights for understanding the mechanism of Mn and Cd uptake in hyperaccumulators and give implications to improve the phytoremediation of Cd-contaminated soil by C. argentea.
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Cadmio , Celosia , Manganeso , Protoplastos , Manganeso/metabolismo , Cadmio/metabolismo , Protoplastos/metabolismo , Celosia/metabolismo , Raíces de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMEN
Heavy metals are dangerous contaminants that constitute a threat to human health because they persist in soils and are easily transferred into the food chain, causing damage to human health. Among heavy metals, nickel appears to be one of the most dangerous, being responsible for different disorders. Public health protection requires nickel detection in the environment and food chains. Biosensors represent simple, rapid, and sensitive methods for detecting nickel contamination. In this paper, we report on the setting up a whole-cell-based system, in which protoplasts, obtained from Nicotiana tabacum leaves, were used as transducers to detect the presence of heavy metal ions and, in particular, nickel ions. Protoplasts were genetically modified with a plasmid containing the Green Fluorescent Protein reporter gene (GFP) under control of the promoter region of a sunflower gene coding for a small Heat Shock Protein (HSP). Using this device, the presence of heavy metal ions was detected. Thus, the possibility of using this whole-cell system as a novel tool to detect the presence of nickel ions in food matrices was assessed.
Asunto(s)
Técnicas Biosensibles , Níquel , Nicotiana , Protoplastos , Níquel/análisis , Protoplastos/metabolismo , Nicotiana/genética , Técnicas Biosensibles/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Contaminación de Alimentos/análisis , Metales Pesados/análisisRESUMEN
CRISPR/Cas9 is widely used for precise mutagenesis through targeted DNA double-strand breaks (DSBs) induction followed by error-prone repair. A better understanding of this process requires measuring the rates of cutting, error-prone, and precise repair, which have remained elusive so far. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterize the dynamics of DSB induction, processing and repair at endogenous loci along a 72 h time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is determined by the combined effect of the rates of DSB induction processing of broken ends, and precise versus error repair. In this study, 64-88% of the molecules were cleaved in the three targets analyzed, while indels ranged between 15-41%. Precise repair accounts for most of the gap between cleavage and error repair, representing up to 70% of all repair events. Altogether, this system exposes flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting the efficiency of CRISPR-mediated mutagenesis.
Asunto(s)
Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , Reparación del ADN , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Edición Génica/métodos , Protoplastos/metabolismo , Mutación INDEL , CinéticaRESUMEN
It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.
Asunto(s)
Daucus carota , Nicotiana , Replicación Viral , Animales , Nicotiana/virología , Nicotiana/microbiología , Daucus carota/virología , Daucus carota/microbiología , Virus ARN/genética , Virus ARN/fisiología , Virus Fúngicos/genética , Virus Fúngicos/clasificación , Virus Fúngicos/fisiología , Filogenia , Protoplastos/virología , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/microbiología , Spodoptera/virología , Spodoptera/microbiologíaRESUMEN
Stress granules (SGs) are conserved cytoplasmic biomolecular condensates mainly formed by proteins and RNA molecules assembled by liquid-liquid phase separation. Isolation of SGs components has been a major challenge in the field due to the dynamic and transient nature of stress granule shells. Here, we describe the methodology for the isolation and visualization of SGs proteins from Arabidopsis thaliana plants using a scaffold component as the target. The protocol consists of the first immunoprecipitation of GFP-tagged scaffold protein, followed by an on-beads enzymatic digestion and previous mass spectrometry identification. Finally, the localization of selected SGs candidates is visualized in Nicotiana benthamiana mesophyll protoplasts.
Asunto(s)
Arabidopsis , Gránulos Citoplasmáticos , Estrés Fisiológico , Arabidopsis/metabolismo , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/química , Proteínas de Arabidopsis/metabolismo , Protoplastos/metabolismo , Nicotiana/metabolismo , Inmunoprecipitación/métodos , Espectrometría de Masas/métodosRESUMEN
Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.
Asunto(s)
Arabidopsis , Proteínas Fluorescentes Verdes , Arabidopsis/virología , Arabidopsis/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas de Transferencia de Gen , Plásmidos/genética , Poliaminas/química , Protoplastos/metabolismo , Nanoestructuras/química , ADN/química , ADN/administración & dosificaciónRESUMEN
KEY MESSAGE: A lipofectamine-mediated transfection protocol for DNA-free genome editing of citrus protoplast cells using a Cas9/gRNA ribonucleoprotein (RNP) complex resulted in the production of transgene free genome edited citrus.
Asunto(s)
Citrus , Edición Génica , Genoma de Planta , Lípidos , Nanopartículas , Ribonucleoproteínas , Edición Génica/métodos , Citrus/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Lípidos/química , Nanopartículas/química , Sistemas CRISPR-Cas , Proteína 9 Asociada a CRISPR/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Protoplastos/metabolismo , Transgenes , Cationes/metabolismo , LiposomasRESUMEN
BACKGROUND: Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas. RESULTS: In the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L- 1), kinetin (KN, 0, 0.5, and 1.0 mg L- 1) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L- 1 2,4-D + 0.5 mg L- 1 KN + 2 nM PSK, 0.5 mg L- 1 2,4-D + 0.9 mg L- 1 KN and 90 nM PSK, and 1.5 mg L- 1 2,4-D and 1 mg L- 1 KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration. CONCLUSIONS: This study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.
Asunto(s)
Angelica , Reguladores del Crecimiento de las Plantas , Técnicas de Embriogénesis Somática de Plantas , Protoplastos , Angelica/embriología , Reguladores del Crecimiento de las Plantas/farmacología , Técnicas de Embriogénesis Somática de Plantas/métodos , Protoplastos/efectos de los fármacos , División Celular/efectos de los fármacosRESUMEN
Plant cells' ability to withstand abiotic stress is strongly linked to modifications in their mechanical characteristics. Nevertheless, the lack of a workable method for consistently tracking plant cells' mechanical properties severely restricts our comprehension of the mechanical alterations in plant cells under stress. In this study, we used the Double Resonator Piezoelectric Cytometry (DRPC) method to dynamically and non-invasively track changes in the surface stress (ΔS) generated and viscoelasticity (storage modulus G' and loss modulus Gâ³) of protoplasts and suspension cells of rice under a drought stress of 5-25% PEG6000. The findings demonstrate that rice suspension cells and protoplasts react mechanically differently to 5-15% PEG6000 stress, implying distinct resistance mechanisms. However, neither of them can withstand 25% PEG6000 stress; they respond mechanically similarly to 25% PEG6000 stress. The results of DRPC are further corroborated by the morphological alterations of rice cells and protoplasts observed under an optical microscope. To sum up, the DRPC technique functions as a precise cellular mechanical sensor and offers novel research tools for the evaluation of plant cell adversity and differentiating between the mechanical reactions of cells and protoplasts under abiotic stress.
Asunto(s)
Oryza , Polietilenglicoles , Protoplastos , Estrés Fisiológico , Sequías , Células VegetalesRESUMEN
Disease resistance gene (R gene)-encoded nucleotide-binding leucine-rich repeat proteins (NLRs) are critical players in plant host defence mechanisms because of their role as receptors that recognise pathogen effectors and trigger plant effector-triggered immunity (ETI). This study aimed to determine the putative role of a cassava coiled-coil (CC)-NLR (CNL) gene MeRPPL1 (Manes.12G091600) (single allele) located on chromosome 12 in the tolerance or susceptibility to South African cassava mosaic virus (SACMV), one of the causal agents of cassava mosaic disease (CMD). A transient protoplast system was used to knock down the expression of MeRPPL1 by clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9). The MeRPPL1-targeting CRISPR vectors and/or SACMV DNA A and DNA B infectious clones were used to transfect protoplasts isolated from leaf mesophyll cells from the SACMV-tolerant cassava (Manihot esculenta) cultivar TME3. The CRISPR/Cas9 silencing vector significantly reduced MeRPPL1 expression in protoplasts whether with or without SACMV co-infection. Notably, SACMV DNA A replication was higher in protoplasts with lower MeRPPL1 expression levels than in non-silenced protoplasts. Mutagenesis studies revealed that protoplast co-transfection with CRISPR-MeRPPL1 silencing vector + SACMV and transfection with only SACMV induced nucleotide substitution mutations that led to altered amino acids in the highly conserved MHD motif of the MeRPPL1-translated polypeptide. This may abolish or alter the regulatory role of the MHD motif in controlling R protein activity and could contribute to the increase in SACMV-DNA A accumulation observed in MeRPPL1-silenced protoplasts. The results herein demonstrate for the first time a role for a CNL gene in tolerance to a geminivirus in TME3.
Asunto(s)
Begomovirus , Manihot , Enfermedades de las Plantas , Proteínas de Plantas , Replicación Viral , Manihot/virología , Manihot/genética , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Begomovirus/genética , Begomovirus/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Geminiviridae/genética , Geminiviridae/fisiología , Sistemas CRISPR-Cas , Resistencia a la Enfermedad/genética , Protoplastos/virología , Protoplastos/metabolismo , Proteínas Repetidas Ricas en LeucinaRESUMEN
BACKGROUND: Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. METHODS: In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. RESULTS: Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. CONCLUSIONS: This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.
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Eucommiaceae , Protoplastos , Transfección , Protoplastos/metabolismo , Eucommiaceae/genética , Eucommiaceae/metabolismo , Transfección/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , PolietilenglicolesRESUMEN
It was previously reported that stress induces a cellular production of abscisic acid in plants, but no direct method shows the evidence. Here, an electrochemical microsensor involving an abscisic acid receptor PYL2 modified carbon fiber microelectrode was fabricated by self-assembly method, where the Cu2+ combined with the histidine tag of PYL2 on the surface of microelectrode was used as the detection probe, the mediated reaction between Cu+ and ferricyanide realized the amplification responses and provided the microsensor with a high sensitivity for detection of abscisic acid with a detection limit of 0.8 nM. With use of this microsensor, an increase of extracellular abscisic acid from single rice protoplast induced by sulfate, osmotic and salinity stress was real-time monitored. Direct measurement of free extracellular abscisic acid in single plant cells might offer important new insights into its role in plants challenged by abiotic stresses.
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Ácido Abscísico , Microelectrodos , Oryza , Proteínas de Plantas , Protoplastos , Oryza/metabolismo , Oryza/química , Ácido Abscísico/metabolismo , Protoplastos/metabolismo , Proteínas de Plantas/metabolismo , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , Cobre/metabolismo , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Ferricianuros/química , Ferricianuros/metabolismoRESUMEN
The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type-V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1-RRA variant did not perform better than the wild-type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants.
Asunto(s)
Edición Génica , Oryza , Solanum lycopersicum , Oryza/genética , Solanum lycopersicum/genética , Sistemas CRISPR-Cas , Protoplastos/metabolismo , Genoma de PlantaRESUMEN
Current techniques identifying herbal medicine species require marker labeling or lack systematical accuracy (expert authentication). There is an emerging interest in developing an accurate and label-free tool for herbal medicine authentication. Here, a high-resolution microfluidic-based method is developed for identifying herbal species by protoplast subpopulations. Moso bamboo and henon bamboo are used as a model to be differentiated based on protoplast. Their biophysical properties factors are characterized to be 7.09 (± 0.39) × 108 V/m2 and 6.54 (± 0.26) × 108 V/m2, respectively. Their biophysical distributions could be distinguished by the Cramér-von Mises criterion with a 94.60% confidence level. The subpopulations of each were compared with conventional flow cytometry indicating the existence of subpopulations and the differences between the two species. The subsets divided by a biophysical factor of 8.05(± 0.51) × 108 V/m2 suggest good consistency with flow cytometry. The work demonstrated the possibility of microfluidics manipulation on protoplast for medication safety use taking advantage of dielectrophoresis. The device is promising in developing a reliable and accurate way of identifying herbal species with difficulties in authentication.
Asunto(s)
Hojas de la Planta , Protoplastos , Análisis de la Célula Individual , Protoplastos/citología , Hojas de la Planta/química , Citometría de Flujo , Técnicas Analíticas Microfluídicas/instrumentación , Microfluídica/instrumentaciónRESUMEN
Plant protoplasts provide starting material for of inducing pluripotent cell masses that are competent for tissue regeneration in vitro, analogous to animal induced pluripotent stem cells (iPSCs). Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterised by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what factors influence chromatin transitions during culturing are largely unknown. Here, we used high-throughput imaging and a custom supervised image analysis protocol extracting over 100 chromatin features of cultured protoplasts. The analysis revealed rapid, multiscale dynamics of chromatin patterns with a trajectory that strongly depended on nutrient availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating intrinsic entropy as a hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.
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Cromatina , Entropía , Células Madre Pluripotentes Inducidas , Cromatina/metabolismo , Cromatina/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Protoplastos/metabolismo , Reprogramación Celular/genética , Histonas/metabolismo , Histonas/genética , Células Vegetales/metabolismo , Epigénesis GenéticaRESUMEN
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.
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Arabidopsis , Hojas de la Planta , Protoplastos , Arabidopsis/metabolismo , Arabidopsis/genética , Protoplastos/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Mapeo de Interacción de Proteínas/métodos , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Microscopía Fluorescente/métodos , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genética , Nicotiana/metabolismo , Nicotiana/genética , Unión Proteica , Agrobacterium/genética , Agrobacterium/metabolismoRESUMEN
Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M. mycetomatis is able to cause this disease. However, the genetic toolbox for M. mycetomatis is limited. To date, no method is available to genetically modify M. mycetomatis. In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M. mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M. mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies.
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Higromicina B , Madurella , Protoplastos , Transformación Genética , Higromicina B/farmacología , Higromicina B/análogos & derivados , Madurella/genética , Madurella/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Micetoma/microbiología , Micetoma/tratamiento farmacológico , Cinamatos/farmacologíaRESUMEN
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.