0 Q&A 325 Views Dec 20, 2022

MicroRNAs (miRNA) are small (21–24 nt) non-coding RNAs involved in many biological processes in both plants and animals. The biogenesis of plant miRNAs starts with the transcription of MIRNA (MIR) genes by RNA polymerase II; then, the primary miRNA transcripts are cleaved by Dicer-like proteins into mature miRNAs, which are then loaded into Argonaute (AGO) proteins to form the effector complex, the miRNA-induced silencing complex (miRISC). In Arabidopsis , some MIR genes are expressed in a tissue-specific manner; however, the spatial patterns of MIR gene expression may not be the same as the spatial distribution of miRISCs due to the non-cell autonomous nature of some miRNAs, making it challenging to characterize the spatial profiles of miRNAs. A previous study utilized protoplasting of green fluorescent protein (GFP) marker transgenic lines followed by fluorescence-activated cell sorting (FACS) to isolate cell-type-specific small RNAs. However, the invasiveness of this approach during the protoplasting and cell sorting may stimulate the expression of stress-related miRNAs. To non-invasively profile cell-type-specific miRNAs, we generated transgenic lines in which root cell layer-specific promoters drive the expression of AGO1 and performed immunoprecipitation to non-invasively isolate cell-layer-specific miRISCs. In this protocol, we provide a detailed description of immunoprecipitation of root cell layer-specific GFP-AGO1 using EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic plants, followed by small RNA sequencing to profile single-cell-type-specific miRNAs. This protocol is also suitable to profile cell-type-specific miRISCs in other tissues or organs in plants, such as flowers or leaves.

Graphical abstract

0 Q&A 486 Views Dec 5, 2022

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall.

0 Q&A 1137 Views Aug 20, 2022

Autophagy is an evolutionarily conserved intracellular degradation process. During autophagy, a set of autophagy-related (ATG) proteins orchestrate the formation of double-bound membrane vesicles called autophagosomes to engulf cytoplasmic material and deliver it to the vacuole for breakdown. Among ATG proteins, the ATG8 is the only one decorating mature autophagosomes and therefore is regarded as a bona fide autophagic marker; colocalization assays with ATG8 are wildly used as a reliable method to identify the components of autophagy machinery or autophagic substrates. Here, we describe a colocalization assay with fluorescent-tagged ATG8 using a tobacco (Nicotiana benthamiana)-based transient expression system.

0 Q&A 2470 Views Jan 20, 2022

The plant nucleus is an important subcellular organelle that contains the genome, ribosomal RNA, and regulatory proteins, and performs a central role in the functioning and metabolism of the cell. Fractionation of intact nuclei is a crucial process to elucidate the function of nuclear proteins. Here, we present a simple method for the fractionation of crude nuclei and extraction of nuclear proteins, based on previously established methods. This protocol provides an easy and quick method to isolate crude nuclei and extract nuclear proteins from Arabidopsis seedlings, which is useful for the research on the nuclear proteins, without requirement for high-purity nuclei.

Graphic abstract:

Schematic procedure for the isolation of crude nuclei and extraction of nuclear proteins from Arabidopsis seedlings.

0 Q&A 2308 Views Nov 20, 2021

Eukaryotic cells use a diverse set of transporters to control the movement of lipids across their plasma membrane, which drastically affects membrane properties. Various tools and techniques to analyze the activity of these transporters have been developed. Among them, assays based on fluorescent phospholipid probes are particularly suitable, allowing for imaging and quantification of lipid internalization in living cells. Classically, these assays have been applied to yeast and animal cells. Here, we describe the adaptation of this powerful approach to characterize lipid internalization in plant roots and aerial tissues using confocal imaging.

Graphic abstract:

Fluorescent lipid uptake in Arabidopsis seedlings. Scale bars: seedling, 25 mm; leaf, 10 μm; root, 25 μm.

0 Q&A 2398 Views Nov 20, 2021

Mesophyll protoplasts freshly isolated from leaves are a useful research system in plants. However, cell walls in woody plants contain more pectin, making mesophyll protoplasts isolation difficult in Populus. This has limited their application in biochemical, molecular, cellular, genetic, genomic, transcriptomic, and proteomic assays. In this protocol, a simple and efficient method to prepare and transfect mesophyll protoplasts of Populus tomentosa is presented in detail. Leaves of P. tomentosa plants grown in tissue culture media were pre-treated in D-mannitol solution and then digested with an enzyme solution. After washing with W5 and MMg buffers, the protoplasts were incubated in PEG/Ca2+ solution with plasmid for transfection. The mesophyll protoplasts isolated were used to express the histone variant H2B fused with green fluorescent protein (GFP) for confocal microscopy imaging. This “P. tomentosa mesophyll protoplasts preparation and transfection” system provides a useful tool for studying woody plants using a variety of applications, including gene expression, subcellular localization, protein-protein interaction, chromatin immunoprecipitation, western blot, single-cell sequencing, and genome editing.

0 Q&A 1907 Views Nov 5, 2021

RNA granules (RGs) are membraneless intracellular compartments that play important roles in the post-transcriptional control of gene expression. Stress granules (SGs) are a type of RGs that form under environmental challenges and/or internal cellular stresses. Stress treatments lead to strong mRNAs translational inhibition and storage in SGs until the normal growth conditions are restored. Intriguingly, we recently showed that plant stress granules are associated with siRNA bodies, where the RDR6-mediated and transposon-derived siRNA biogenesis occurs (Kim et al., 2021). This protocol provides a technical workflow for the enrichment of cytoplasmic RGs from Arabidopsis seedlings. We used the DNA methylation-deficient ddm1 mutant in our study, but the method can be applied to any other plant samples with strong RG formation. The resulting RG fractions can be further tested for either RNAs or proteins using RNA-seq and mass spectrometry-based proteomics.

0 Q&A 1599 Views Oct 20, 2021

Defense priming describes the enhanced potency of cells to activate defense responses. Priming accompanies local and systemic immune responses and can be triggered by microbial infection or upon treatment with certain chemicals. Thus, chemically activating defense priming is promising for biomedicine and agriculture. However, test systems for spotting priming-inducing chemicals are rare. Here, we describe a high-throughput screen for compounds that prime microbial pattern-spurred secretion of antimicrobial furanocoumarins in parsley culture cells. For the best possible throughput, we perform the assay with 1-ml aliquots of cell culture in 24-well microtiter plates. The advantages of the non-invasive test over competitive assays are its simplicity, remarkable reliability, and high sensitivity, which is based on furanocoumarin fluorescence in UV light.

0 Q&A 2478 Views Aug 20, 2021

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers.

Graphic abstract:

Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.

0 Q&A 2652 Views Jun 20, 2021

Plant nanobiotechnology is a flourishing field that uses nanomaterials to study and engineer plant function. Applications of nanotechnology in plants have great potential as tools for improving crop yield, tolerance to disease and environmental stress, agrochemical delivery of pesticides and fertilizers, and genetic modification and transformation of crop plants. Previous studies have used nanomaterials functionalized with chemicals, including biocompatible polymers with charged, neutral, or hydrophobic functional groups, to improve nanomaterial uptake and localization in plant cells. Recently, the use of biorecognition motifs such as peptides has been demonstrated to enable the targeted delivery of nanoparticles in plants (Santana et al., 2020). Herein, we describe a bio-protocol to target nanoparticles with chemical cargoes to chloroplasts in plant leaves and assess targeting efficiency using advanced analytical tools, including confocal microscopy and elemental analysis. We also describe the use of isothermal titration calorimetry to determine the affinity of nanomaterials for their chemical cargoes. Nanotechnology-based methods for targeted delivery guided by conserved plant molecular recognition mechanisms will provide more robust plant bioengineering tools across diverse plant species.

Graphic abstract:

Targeted delivery of nanomaterials with chemical cargoes to chloroplasts enabled by plant biorecognition