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Gene Jun 2024Protocatechualdehyde is a plant natural phenolic aldehyde and an active ingredient with important bioactivities in traditional Chinese medicine. Protocatechualdehyde is...
Protocatechualdehyde is a plant natural phenolic aldehyde and an active ingredient with important bioactivities in traditional Chinese medicine. Protocatechualdehyde is also a key intermediate in the synthesis of Amaryllidaceae alkaloids for supplying the C6-C1 skeleton. However, the biosynthesis of protocatechualdehyde in plants remains obscure. In this study, we measured the protocatechualdehyde contents in the root, bulb, scape and flower of the Amaryllidaceae plant Lycoris aurea (L'Hér.) Herb., and performed the correlation analysis between the protocatechualdehyde contents and the transcriptional levels of the phenolic oxidization candidate protein encoding genes. We found that a novel ascorbate peroxidase encoded by the contig_24999 in the L. aurea transcriptome database had potential role in the biosynthesis of protocatechualdehyde. The LauAPX_24999 gene was then cloned from the cDNA of the scape of L. aurea. The transient expression of LauAPX_24999 protein in Arabidopsis protoplasts demonstrated that LauAPX_24999 protein was localized in the cytoplasm, thus belonging to Class II L-ascorbate peroxidase. Subsequently, LauAPX_24999 protein was heterogenously expressed in Escherichia coli, and identified that LauAPX_24999 biosynthesized protocatechualdehyde from p-hydroxybenzaldehyde using L-ascorbic acid as the electron donor. The protein structure modelling and molecular docking indicated that p-hydroxybenzaldehyde could access to the active pocket of LauAPX_24999 protein, and reside at the δ-edge of the heme group while L-ascorbic acid binds at the γ-heme edge. To our knowledge, LauAPX_24999 is the first enzyme discovered in plants able to biosynthesize protocatechualdehyde from p-hydroxybenzaldehyde, and offers a competent enzyme resource for the biosynthesis of Amaryllidaceae alkaloids via synthetic biology.
PubMed: 38880186
DOI: 10.1016/j.gene.2024.148697 -
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 -
Plant Cell Reports Jun 2024A lipofectamine-mediated transfection protocol for DNA-free genome editing of citrus protoplast cells using a Cas9/gRNA ribonucleoprotein (RNP) complex resulted in the...
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.
Topics: Gene Editing; Citrus; Ribonucleoproteins; Genome, Plant; Lipids; Nanoparticles; CRISPR-Cas Systems; CRISPR-Associated Protein 9; RNA, Guide, CRISPR-Cas Systems; Protoplasts; Transgenes; Cations; Liposomes
PubMed: 38874819
DOI: 10.1007/s00299-024-03254-3 -
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 -
Methods in Molecular Biology (Clifton,... 2024Stress granules (SGs) are conserved cytoplasmic biomolecular condensates mainly formed by proteins and RNA molecules assembled by liquid-liquid phase separation....
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.
Topics: Arabidopsis; Cytoplasmic Granules; Stress, Physiological; Arabidopsis Proteins; Protoplasts; Nicotiana; Immunoprecipitation; Mass Spectrometry
PubMed: 38869787
DOI: 10.1007/978-1-0716-3973-3_4 -
Proceedings of the National Academy of... Jun 2024It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been...
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 could cross multiple kingdoms. 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 fungus could replicate in protoplasts of the carrot (), and , 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 and the fruit fly . Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.
Topics: Animals; Nicotiana; Virus Replication; Daucus carota; RNA Viruses; Fungal Viruses; Phylogeny; Protoplasts; Plant Diseases; Spodoptera
PubMed: 38865269
DOI: 10.1073/pnas.2318150121 -
The Plant Journal : For Cell and... Jun 2024Strigolactones (SL) function as plant hormones in control of multiple aspects of plant development, mostly via the regulation of gene expression. Immediate early-gene...
Strigolactones (SL) function as plant hormones in control of multiple aspects of plant development, mostly via the regulation of gene expression. Immediate early-gene regulation by SL remains unexplored due to difficulty in dissecting early from late gene expression responses to SL. We used synthetic SL, rac-GR24 treatment of protoplasts and RNA-seq to explore early SL-induced changes in gene expression over time (5-180 minutes) and discovered rapid, dynamic and SL receptor D14-dependent regulation of gene expression in response to rac-GR24. Importantly, we discovered a significant dependence of SL signalling on chromatin remodelling processes, as the induction of a key SL-induced transcription factor BRANCHED1 requires the SWI/SNF chromatin remodelling ATPase SPLAYED (SYD) and leads to upregulation of a homologue SWI/SNF ATPase BRAHMA. ATAC-seq profiling of genome-wide changes in chromatin accessibility in response to rac-GR24 identified large-scale changes, with over 1400 differentially accessible regions. These changes in chromatin accessibility often precede transcriptional changes and are likely to harbour SL cis-regulatory elements. Importantly, we discovered that this early and extensive modification of the chromatin landscape also requires SYD. This study, therefore, provides evidence that SL signalling requires regulation of chromatin accessibility, and it identifies genomic locations harbouring likely SL cis-regulatory sequences.
PubMed: 38858857
DOI: 10.1111/tpj.16873 -
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