Plant Science

Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide small RNAs designed to induce highly specific and efficient gene silencing in plants. Traditional approaches rely on the transgenic expression of ~1 kb TAS precursors, which limits their use in non-model species, under strict GMO regulations, and in size-constrained expression or delivery systems. This protocol describes a rapid workflow for the design, assembly, and delivery of syn-tasiRNAs derived from much shorter precursors, referred to as minimal precursors. The pipeline includes in silico design of highly specific syn-tasiRNA sequences, cloning of minimal precursors into plant expression or potato virus X (PVX)-based viral vectors through Golden Gate or Gibson assembly, and delivery to plants through Agrobacterium-mediated expression or by spraying crude extracts containing recombinant PVX expressing the minimal precursors. These methodologies make syn-tasiRNA-based tools more accessible and broadly applicable for plant research and biotechnology across diverse species and experimental contexts.

Plant embryos are contained within seeds. Isolating them is crucial when endosperm and seed coat tissues interfere with the study of mutant genetic functions due to differing genotypes between maternal and embryonic tissues. RNA extraction from plant embryonic tissue presents particular challenges due to the high activity of RNases, the composition of the seed, and the risk of RNA degradation. The developmental stage of the embryo is a key aspect of successful isolation and RNA extraction due to the size and amount of tissue. Proper handling during RNA extraction is critical to maintain RNA integrity and prevent degradation. While commercial kits offer various methods for RNA extraction from embryos, homemade protocols provide valuable advantages, including cost-effectiveness and accessibility for labs with limited funding. Here, we present a simple and efficient protocol for extracting RNA from isolated Arabidopsis thaliana embryos at the torpedo/cotyledon stage using a homemade extraction buffer previously reported for styles of Nicotiana alata.

For obtaining insights into gene networks during plant reproductive development, having transcriptomes of specific cells from developmental stages as starting points is very useful. During development, there is a balance between cell proliferation and differentiation, and many cell and tissue types are formed. While there is a wealth of transcriptome data available, it is mostly at the organ level and not at specific cell or tissue type level. Therefore, methods to isolate specific cell and tissue types are needed. One method is fluorescent activated cell sorting (FACS), but it has limitations such as requiring marker lines and protoplasting. Recently, single-cell/nuclei isolation methods have been developed; however, a minimum amount of genetic information (marker genes) is needed to annotate/predict the resulting cell clusters in these experiments. Another technique that has been known for some time is laser-assisted microdissection (LAM), where specific cells are microdissected and collected using a laser mounted on a microscope platform. This technique has advantages over the others because no fluorescent marker lines must be made, no marker genes must be known, and no protoplasting must be done. The LAM technique consists in tissue fixation, tissue embedding and sectioning using a microtome, microdissection and collection of the cells of interest on the microscope, and finally RNA extraction, library preparation, and RNA sequencing. In this protocol, we implement the use of normal slides instead of the membrane slides commonly used for LAM. We applied this protocol to obtain the transcriptomes of specific tissues during the development of the gynoecium of Arabidopsis.

Here, we present an approach combining fluorescence in situ hybridization (FISH) and immunolabeling for localization of pri-miRNAs in isolated nuclei of A. thaliana. The presented method utilizes specific DNA oligonucleotide probes, modified by addition of digoxigenin-labeled deoxynucleotides to its 3′ hydroxyl terminus by terminal deoxynucleotidyl transferase (TdT). The probes are then detected by immunolabeling of digoxigenin (DIG) using specific fluorescent-labeled antibodies to visualize hybridized probes. Recently, we have applied this method to localize pri-miRNA156a, pri-miRNA163, pri-miRNA393a, and pri-miRNA414 in the nuclei isolated from leaves of 4-week-old A. thaliana. The present approach can be easily implemented to analyze nuclear distribution of diverse RNA classes, including mRNAs and pri-miRNAs in isolated fixed cells or nuclei from plant.

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

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.

Cytidine-to-uridine (C-to-U) RNA editing is one of the most important post-transcriptional RNA processing in plant mitochondria and chloroplasts. Several techniques have been developed to detect the RNA editing efficiency in plant mitochondria and chloroplasts, such as poisoned primer extension (PPE) assays, high-resolution melting (HRM) analysis, and DNA sequencing. Here, we describe a method for the quantitative detection of RNA editing at specific sites by sequencing cDNA from plant leaves to further evaluate the effect of different treatments or plant mutants on the C to U RNA editing in mitochondria and chloroplasts.

Small nuclear RNAs (snRNAs) are vital for eukaryotic cell activities and play important roles in pre-mRNA splicing. The molecular mechanism underlying the transcription of snRNA, regulated via upstream/downstream cis-elements and relevant trans-elements, has been investigated in detail using cell-free extracts. However, the processing of precursor snRNA (pre-snRNA), which is required by 3’ end maturation of pre-snRNA, remains unclear as a proper processing assay is difficult to develop in vitro. Here, we present an in vitro method using synthetic labeled RNA as substrates to study the 3’ cleavage of pre-snRNA.

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.

The micrografting technique in the model plant Arabidopsis has been widely used in the field of plant science. Grafting experiments have demonstrated that signal transductions are systematically regulated in many plant characteristics, including defense mechanisms and responses to surrounding environments such as soil and light conditions. Hypocotyl micrografting is a powerful tool for the analysis of signal transduction between shoots and roots; however, the requirement for a high level of skill for micrografting, during which small seedlings are microdissected and micromanipulated, has limited its use. Here, we developed a silicone-made microdevice, called a micrografting chip, to perform Arabidopsis micrografting easily and uniformly. The micrografting chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path to hold hypocotyl. All micrografting procedures are performed on the chip. This method using a micrografting chip will avoid the need for training and promote studies of systemic signaling in plants.

Graphic abstract:

A silicone chip for easy grafting