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Cell Jun 2021Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a...
Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.
Topics: Animals; Arabidopsis; Arabidopsis Proteins; Calcium; Carrier Proteins; Cell Death; Cell Membrane; Cell Membrane Permeability; Disease Resistance; Glutamic Acid; Lipid Bilayers; Oocytes; Plant Cells; Plant Immunity; Protein Multimerization; Protoplasts; Reactive Oxygen Species; Signal Transduction; Single Molecule Imaging; Vacuoles; Xenopus
PubMed: 33984278
DOI: 10.1016/j.cell.2021.05.003 -
BMC Plant Biology Nov 2014To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant...
BACKGROUND
To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.
RESULTS
We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.
CONCLUSIONS
We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.
Topics: Agrobacterium; Arabidopsis; Base Sequence; CRISPR-Cas Systems; Genetic Engineering; Genetic Vectors; Genome, Plant; Plants, Genetically Modified; Protoplasts; Sequence Alignment; Zea mays
PubMed: 25432517
DOI: 10.1186/s12870-014-0327-y -
Molecular Plant Jan 2021The rapid and enthusiastic adoption of single-cell RNA sequencing (scRNA-seq) has demonstrated that this technology is far more than just another way to perform... (Review)
Review
The rapid and enthusiastic adoption of single-cell RNA sequencing (scRNA-seq) has demonstrated that this technology is far more than just another way to perform transcriptome analysis. It is not an exaggeration to say that the advent of scRNA-seq is revolutionizing the details of whole-transcriptome snapshots from a tissue to a cell. With this disruptive technology, it is now possible to mine heterogeneity between tissue types and within cells like never before. This enables more rapid identification of rare and novel cell types, simultaneous characterization of multiple different cell types and states, more accurate and integrated understanding of their roles in life processes, and more. However, we are only at the beginning of unlocking the full potential of scRNA-seq applications. This is particularly true for plant sciences, where single-cell transcriptome profiling is in its early stage and has many exciting challenges to overcome. In this review, we compare and evaluate recent pioneering studies using the Arabidopsis root model, which has established new paradigms for scRNA-seq studies in plants. We also explore several new and promising single-cell analysis tools that are available to those wishing to study plant development and physiology at unprecedented resolution and scale. In addition, we propose some future directions on the use of scRNA-seq technology to tackle some of the critical challenges in plant research and breeding.
Topics: Cell Size; Gene Expression Profiling; Genomics; Plants; Protoplasts; Single-Cell Analysis
PubMed: 33152518
DOI: 10.1016/j.molp.2020.10.012 -
Proceedings of the National Academy of... Mar 2015The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome...
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.
Topics: Base Sequence; CRISPR-Cas Systems; Genes, Plant; Genetic Engineering; Molecular Sequence Data; Mutagenesis; Mutation; Oryza; Plants, Genetically Modified; Protoplasts; RNA Editing; RNA Processing, Post-Transcriptional; RNA, Guide, CRISPR-Cas Systems; RNA, Transfer
PubMed: 25733849
DOI: 10.1073/pnas.1420294112 -
Developmental Cell Feb 2021Crop productivity depends on activity of meristems that produce optimized plant architectures, including that of the maize ear. A comprehensive understanding of...
Crop productivity depends on activity of meristems that produce optimized plant architectures, including that of the maize ear. A comprehensive understanding of development requires insight into the full diversity of cell types and developmental domains and the gene networks required to specify them. Until now, these were identified primarily by morphology and insights from classical genetics, which are limited by genetic redundancy and pleiotropy. Here, we investigated the transcriptional profiles of 12,525 single cells from developing maize ears. The resulting developmental atlas provides a single-cell RNA sequencing (scRNA-seq) map of an inflorescence. We validated our results by mRNA in situ hybridization and by fluorescence-activated cell sorting (FACS) RNA-seq, and we show how these data may facilitate genetic studies by predicting genetic redundancy, integrating transcriptional networks, and identifying candidate genes associated with crop yield traits.
Topics: Base Sequence; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Gene Regulatory Networks; Genetic Association Studies; Protoplasts; Quantitative Trait Loci; Reproducibility of Results; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome; Zea mays
PubMed: 33400914
DOI: 10.1016/j.devcel.2020.12.015 -
Plant Physiology Apr 2019Single-cell RNA sequencing (scRNA-seq) has been used extensively to study cell-specific gene expression in animals, but it has not been widely applied to plants. Here,...
Single-cell RNA sequencing (scRNA-seq) has been used extensively to study cell-specific gene expression in animals, but it has not been widely applied to plants. Here, we describe the use of a commercially available droplet-based microfluidics platform for high-throughput scRNA-seq to obtain single-cell transcriptomes from protoplasts of more than 10,000 Arabidopsis () root cells. We find that all major tissues and developmental stages are represented in this single-cell transcriptome population. Further, distinct subpopulations and rare cell types, including putative quiescent center cells, were identified. A focused analysis of root epidermal cell transcriptomes defined developmental trajectories for individual cells progressing from meristematic through mature stages of root-hair and nonhair cell differentiation. In addition, single-cell transcriptomes were obtained from root epidermis mutants, enabling a comparative analysis of gene expression at single-cell resolution and providing an unprecedented view of the impact of the mutated genes. Overall, this study demonstrates the feasibility and utility of scRNA-seq in plants and provides a first-generation gene expression map of the Arabidopsis root at single-cell resolution.
Topics: Arabidopsis; Feasibility Studies; Plant Epidermis; Plant Roots; Protoplasts; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome
PubMed: 30718350
DOI: 10.1104/pp.18.01482 -
Cytometry. Part a : the Journal of the... Apr 2021
Topics: Flow Cytometry; Protoplasts
PubMed: 33398930
DOI: 10.1002/cyto.a.24295 -
Microorganisms Feb 2023Single-cell sequencing (SCS) is an evolutionary technique for conducting life science research, providing the highest genome-sale throughput and single-cell resolution... (Review)
Review
Single-cell sequencing (SCS) is an evolutionary technique for conducting life science research, providing the highest genome-sale throughput and single-cell resolution and unprecedented capabilities in addressing mechanistic and operational questions. Unfortunately, the current SCS pipeline cannot be directly applied to algal research as algal cells have cell walls, which makes RNA extraction hard for the current SCS platforms. Fortunately, effective methods are available for producing algal protoplasts (cells without cell walls), which can be directly fed into current SCS pipelines. In this review, we first summarize the cell wall structure and chemical composition of algal cell walls, particularly in Chlorophyta, then summarize the advances made in preparing algal protoplasts using physical, chemical, and biological methods, followed by specific cases of algal protoplast production in some commonly used eukaryotic algae. This review provides a timely primer to those interested in applying SCS in eukaryotic algal research.
PubMed: 36838504
DOI: 10.3390/microorganisms11020538 -
Plants (Basel, Switzerland) Feb 2023Species of the family Apiaceae occupy a major market share but are hitherto dependent on open pollinated cultivars. This results in a lack of production uniformity and... (Review)
Review
Species of the family Apiaceae occupy a major market share but are hitherto dependent on open pollinated cultivars. This results in a lack of production uniformity and reduced quality that has fostered hybrid seed production. The difficulty in flower emasculation led breeders to use biotechnology approaches including somatic hybridization. We discuss the use of protoplast technology for the development of somatic hybrids, cybrids and in-vitro breeding of commercial traits such as CMS (cytoplasmic male sterility), GMS (genetic male sterility) and EGMS (environment-sensitive genic male sterility). The molecular mechanism(s) underlying CMS and its candidate genes are also discussed. Cybridization strategies based on enucleation (Gamma rays, X-rays and UV rays) and metabolically arresting protoplasts with chemicals such as iodoacetamide or iodoacetate are reviewed. Differential fluorescence staining of fused protoplast as routinely used can be replaced by new tagging approaches using non-toxic proteins. Here, we focused on the initial plant materials and tissue sources for protoplast isolation, the various digestion enzyme mixtures tested, and on the understanding of cell wall re-generation, all of which intervene in somatic hybrids regeneration. Although there are no alternatives to somatic hybridization, various approaches also discussed are emerging, viz., robotic platforms, artificial intelligence, in recent breeding programs for trait identification and selection.
PubMed: 36903923
DOI: 10.3390/plants12051060 -
Frontiers in Genome Editing 2021In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating... (Review)
Review
In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.
PubMed: 34713263
DOI: 10.3389/fgeed.2021.717017