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Horticulture Research Jan 2024CRISPR-Cas technologies allow for precise modifications in plant genomes and promise to revolutionize agriculture. These technologies depend on the delivery of editing...
CRISPR-Cas technologies allow for precise modifications in plant genomes and promise to revolutionize agriculture. These technologies depend on the delivery of editing components into plant cells and the regeneration of fully edited plants. In vegetatively propagated plants, such as grape, protoplast culture provides one of the best avenues for producing non-chimeric and transgene-free genome-edited plants. However, poor regeneration of plants from protoplasts has hindered their implementation for genome editing. Here, we report an efficient protocol for regenerating plants from protoplasts from multiple grape varieties. By encapsulating the protoplasts in calcium alginate beads and co-culturing them with feeder cultures, the protoplasts divide to form callus colonies that regenerate into embryos and ultimately plants. This protocol worked successfully in wine and table grape () varieties, as well as grape rootstocks and the grapevine wild relative . Moreover, by transfecting protoplasts with CRISPR-plasmid or ribonucleoprotein (RNP) complexes, we regenerated albino plants with edits in gene in three varieties and in . The results reveal the potential of this platform to facilitate genome editing in species.
PubMed: 38895602
DOI: 10.1093/hr/uhad266 -
International Journal of Molecular... May 2024Heavy 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...
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 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 reporter gene () 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.
Topics: Nickel; Protoplasts; Nicotiana; Biosensing Techniques; Green Fluorescent Proteins; Food Contamination; Metals, Heavy
PubMed: 38892274
DOI: 10.3390/ijms25116090 -
International Journal of Molecular... May 2024Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While and rice have been extensively...
Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While and rice have been extensively studied, research on foxtail millet is limited. This study identified two transcripts of , both of which were induced by NO and showed similar expression patterns. The overexpression of and in inhibited plant growth and seed size, although SiAAP9 was found to transport more amino acids into seeds. Furthermore, transgenic showed increased tolerance to high concentrations of glutamate (Glu) and histidine (His). The high overexpression level of suggested its protein was not only located on the plasma membrane but potentially on other organelles, as well. Interestingly, sequence deletion reduced SiAAP9's sensitivity to Brefeldin A (BFA), and SiAAP9 had ectopic localization on the endoplasmic reticulum (ER). Protoplast amino acid uptake experiments indicated that SiAAP9 enhanced Glu transport into foxtail millet cells. Overall, the two transcripts of have similar functions, but SiAAP9L shows a higher colocalization with BFA compartments compared to SiAAP9S. Our research identifies a potential candidate gene for enhancing the nutritional quality of foxtail millet through breeding.
Topics: Arabidopsis; Plants, Genetically Modified; Gene Expression Regulation, Plant; Plant Proteins; Endoplasmic Reticulum; Seeds; Setaria Plant; Amino Acid Transport Systems; Protein Transport; Brefeldin A; Amino Acids; Glutamic Acid
PubMed: 38892028
DOI: 10.3390/ijms25115840 -
Nature Communications Jun 2024CRISPR/Cas9 is widely used for precise mutagenesis through targeted DNA double-strand breaks (DSBs) induction followed by error-prone repair. A better understanding of...
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.
Topics: DNA Breaks, Double-Stranded; CRISPR-Cas Systems; DNA Repair; Solanum lycopersicum; Gene Editing; Protoplasts; INDEL Mutation; Kinetics
PubMed: 38877047
DOI: 10.1038/s41467-024-49410-x -
Frontiers in Microbiology 2024Ganoderic acids (GAs) are major functional components of . The study aimed to breed a new strain with increased contents of individual GAs. Two mating-compatible...
Ganoderic acids (GAs) are major functional components of . The study aimed to breed a new strain with increased contents of individual GAs. Two mating-compatible monokaryotic strains, G. 260125 and G. 260124, were successfully isolated from the dikaryotic CGMCC 5.0026 via protoplast formation and regeneration. The hemoglobin gene () and squalene synthase gene () were overexpressed in the monokaryotic G. 260124 and G. 260125 strain, respectively. Mating between the G. 260124 strain overexpressing and the G. 260125 strain overexpressing sqs resulted in the formation of the new hybrid dikaryotic strain sqs-vgb. The maximum contents of ganoderic acid (GA)-T, GA-Me, and GA-P in the fruiting body of the mated sqs-vgb strain were 23.1, 15.3, and 39.8 μg/g dry weight (DW), respectively, 2.23-, 1.75-, and 2.69-fold greater than those in 5.0026. The squalene and lanosterol contents increased 2.35- and 1.75-fold, respectively, in the fruiting body of the mated sqs-vgb strain compared with those in the 5.0026. In addition, the maximum expression levels of the and lanosterol synthase gene () were increased 3.23- and 2.13-fold, respectively, in the mated sqs-vgb strain. In summary, we developed a new strain with higher contents of individual GAs in the fruiting body by integrating genetic engineering and mono-mono crossing.
PubMed: 38873146
DOI: 10.3389/fmicb.2024.1410368 -
Microbiological Research Jun 2024Plants have developed intricate immune mechanisms to impede Phytophthora colonization. In response, Phytophthora secretes RxLR effector proteins that disrupt plant...
Plants have developed intricate immune mechanisms to impede Phytophthora colonization. In response, Phytophthora secretes RxLR effector proteins that disrupt plant defense and promote infection. The specific molecular interactions through which Phytophthora RxLR effectors undermine plant immunity, however, remain inadequately defined. In this study, we delineate the role of the nuclear-localized RxLR effector PcAvh87, which is pivotal for the full virulence of Phytophthora cinnamomi. Gene expression analysis indicates that PcAvh87 expression is significantly upregulated during the initial infection stages, interacting with the immune responses triggered by the elicitin protein INF1 and pro-apoptotic protein BAX. Utilizing PEG/CaCl-mediated protoplast transformation and CRISPR/Cas9-mediated gene editing, we generated PcAvh87 knockout mutants, which demonstrated compromised hyphal growth, sporangium development, and zoospore release, along with a marked reduction in pathogenicity. This underscores PcAvh87's crucial role as a virulence determinant. Notably, PcAvh87, conserved across the Phytophthora genus, was found to modulate the activity of plant immune protein 113, thereby attenuating plant immune responses. This implies that the PcAvh87-mediated regulatory mechanism could be a common strategy in Phytophthora species to manipulate plant immunity. Our findings highlight the multifaceted roles of PcAvh87 in promoting P. cinnamomi infection, including its involvement in sporangia production, mycelial growth, and the targeting of plant immune proteins to enhance pathogen virulence.
PubMed: 38870619
DOI: 10.1016/j.micres.2024.127789 -
BMC Plant Biology Jun 2024Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially...
Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai.
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), kinetin (KN, 0, 0.5, and 1.0 mg L) 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 2,4-D + 0.5 mg L KN + 2 nM PSK, 0.5 mg L 2,4-D + 0.9 mg L KN and 90 nM PSK, and 1.5 mg L 2,4-D and 1 mg L 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.
Topics: Angelica; Plant Growth Regulators; Plant Somatic Embryogenesis Techniques; Protoplasts; Cell Division
PubMed: 38858674
DOI: 10.1186/s12870-024-05243-w -
The Plant Genome Jun 2024Seven in absentia proteins, which contain a conserved SINA domain, are involved in regulating various aspects of wheat (Triticum aestivum L.) growth and development,...
Seven in absentia proteins, which contain a conserved SINA domain, are involved in regulating various aspects of wheat (Triticum aestivum L.) growth and development, especially in response to environmental stresses. However, it is unclear whether TaSINA family members are involved in regulating grain development until now. In this study, the expression pattern, genomic polymorphism, and relationship with grain-related traits were analyzed for all TaSINA members. Most of the TaSINA genes identified showed higher expression levels in young wheat spikes or grains than other organs. The genomic polymorphism analysis revealed that at least 62 TaSINA genes had different haplotypes, where the haplotypes of five genes were significantly correlated with grain-related traits. Kompetitive allele-specific PCR markers were developed to confirm the single nucleotide polymorphisms in TaSINA101 and TaSINA109 among the five selected genes in a set of 292 wheat accessions. The TaSINA101-Hap II and TaSINA109-Hap II haplotypes had higher grain weight and width compared to TaSINA101-Hap I and TaSINA109-Hap I in at least three environments, respectively. The qRT-PCR assays revealed that TaSINA101 was highly expressed in the palea shell, seed coat, and embryo in young wheat grains. The TaSINA101 protein was unevenly distributed in the nucleus when transiently expressed in the protoplast of wheat. Three homozygous TaSINA101 transgenic lines in rice (Oryza sativa L.) showed higher grain weight and size compared to the wild type. These findings provide valuable insight into the biological function and elite haplotype of TaSINA family genes in wheat grain development at a genomic-wide level.
PubMed: 38840306
DOI: 10.1002/tpg2.20480 -
Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression.Fungal Biology and Biotechnology Jun 2024Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other...
BACKGROUND
Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains.
RESULTS
The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy.
CONCLUSION
The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.
PubMed: 38824542
DOI: 10.1186/s40694-024-00175-4 -
Frontiers in Plant Science 2024Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR)...
Protein disulfide isomerase-9 interacts with the lumenal region of the transmembrane endoplasmic reticulum stress sensor kinase, IRE1, to modulate the unfolded protein response in .
Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR) senses ER stress and restores proteostasis by increasing the expression of ER-resident protein folding chaperones, such as protein disulfide isomerases (PDIs). In plants, the transmembrane ER stress sensor kinase, IRE1, activates the UPR by unconventionally splicing the mRNA encoding the bZIP60 transcription factor, triggering UPR gene transcription. The induced PDIs catalyze disulfide-based polypeptide folding to restore the folding capacity in the ER; however, the substrates with which PDIs interact are largely unknown. Here, we demonstrate that the PDI-M subfamily member, PDI9, modulates the UPR through interaction with IRE1. This PDI9-IRE1 interaction was largely dependent on Cys63 in the first dithiol redox active domain of PDI9, and Cys233 and Cys107 in the ER lumenal domain of IRE1A and IRE1B, respectively. and , PDI9 coimmunoprecipitated with IRE1A and IRE1B. Moreover, the PDI9:RFP and Green Fluorescence Protein (GFP):IRE1 fusions exhibited strong interactions as measured by fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) when coexpressed in mesophyll protoplasts. The UPR-responsive PDI9 promoter:mCherry reporter and the UPR-dependent splicing of the bZIP60 intron from the mRNA of the 35S::bZIP60-intron:GFP reporter were both significantly induced in the mutants, indicating a derepression and hyperactivation of UPR. The inductions of both reporters were substantially attenuated in the mutant. We propose a model in which PDI9 modulates the UPR through two competing activities: secretory protein folding and via interaction with IRE1 to maintain proteostasis in plants.
PubMed: 38817940
DOI: 10.3389/fpls.2024.1389658