0 Q&A 1099 Views Jun 5, 2022

Plant genomes are pronouncedly enriched in repeat elements such as transposons. These repeats are epigenetically regulated by DNA methylation. Whole genome high-depth sequencing after bisulfite treatment remains an expensive and laborious method to reliably profile the DNA methylome, especially when considering large genomes such as in crops. Here, we present a simple reproducible Southern hybridisation–based assay to obtain incontrovertible methylation patterns from targeted regions in the rice genome. By employing minor but key modifications, we reliably detected transposon copy number variations over multiple generations. This method can be regarded as a gold standard for validation of epigenetic variations at target loci, and the consequent proliferation of transposons, or segregation in several plant replicates and genotypes.

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0 Q&A 980 Views Apr 20, 2022

Biotin is an essential vitamin in plants. However, characterization of biotin deficiency has been limited by embryo lethality in mutants, which can only be rescued by supplementation of biotin. Here, we describe a protocol to characterize biotin deficiency in Arabidopsis thaliana through application of the polyamine cadaverine. Cadaverine induces changes in primary root growth. Protein biotinylation in Arabidopsis seedlings can be quantified through an assay similar to a western blot, in which protein biotinylation is detected by a streptavidin probe. This technique provides a chemical means of inhibiting biotin synthesis, allowing for further characterization of biotin deficiency on a physiological and molecular level.

0 Q&A 2395 Views Dec 5, 2021

Gene expression depends on the binding of transcription factors with DNA regulatory sequences. The level of accessibility for these sequences varies between cells and cell types. Until recently, using the Tn5 assay for transposase-accessible chromatin for sequencing (ATAC-seq) technology allowed assessing the profiles of chromatin from an entire organ or, when coupled with the isolation of nuclei tagged in specific cell types (INTACT) method, from a cell-type. Recently, ATAC-seq experiments were conducted at the level of individual plant nuclei. Applying single nuclei ATAC-seq (sNucATAC-seq) technology to thousands of individual cells revealed more finely tuned profiles of chromatin accessibility. In this manuscript, we describe a method to isolate nuclei fom plant roots and green tissues, permeabilize the nuclear membrane using detergent to allow the penetration of the Tn5 transposase, and re-suspend them in a nuclei resuspension buffer compatible with the construction of sNucATAC-seq libraries using the 10× Genomic’s Chromium technology. This protocol was successfully applied on Arabidopsis thaliana and Glycine max root nuclei.

0 Q&A 3394 Views Dec 5, 2021

The experimental identification of protein-protein interactions (PPIs) is critical to understand protein function. Thus, a plethora of sensitive and versatile approaches have been developed to detect PPIs in vitro or in vivo, such as protein pull-down, yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP), and bimolecular fluorescence complementation (BiFC) assays. The recently established split-luciferase complementation (Split-LUC) imaging assay has several advantages compared to other approaches to detect PPIs in planta: it is a relatively simple and fast method to detect PPIs in vivo; the results are quantitative, with high sensitivity and low background; it measures dynamic PPIs in real-time; and it requires limited experimental materials and instrumentation. In this assay, the amino-terminal and carboxyl-terminal halves of the luciferase enzyme are fused to two proteins of interest (POIs), respectively; the luciferase protein is reconstituted when two POIs interact with each other, giving rise to a measurable activity. Here, we describe a protocol for the Split-LUC imaging assay using a pair of modified gateway-compatible vectors upon Agrobacterium-mediated transient expression in Nicotiana benthamiana. With this setup, we have successfully confirmed a series of interactions among virus-plant proteins, virus-virus proteins, plant-plant proteins, or bacteria-plant proteins in N. benthamiana.

0 Q&A 1887 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.

1 Q&A 2560 Views Nov 5, 2021

Characterizing the molecular mechanisms regulating gene expression is crucial for understanding the regulatory processes underlying physiological responses to environmental and developmental signals in eukaryotes. The covalent modification of histones contributes to the compaction levels of chromatin, as well as the recruitment of the transcriptional machinery to specific loci, facilitating metastable changes in gene activity. ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) has become the gold standard method for determining histone modification profiles among different organisms, tissues, and genotypes. In the current protocol, we describe a highly robust method for performing ChIP-seq of histone modifications in Arabidopsis thaliana plantlets. Besides its robustness, this method uses in-house-prepared buffers for chromatin extraction, immunoprecipitation, washing, and elusion, making it cost-effective in contrast to commercial kits.

0 Q&A 1714 Views Sep 20, 2021

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.

0 Q&A 1290 Views Sep 5, 2021

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.

0 Q&A 2455 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 2599 Views Aug 5, 2021

Ralstonia solanacearum is a devastating soil-borne bacterial pathogen that causes disease in multiple host plants worldwide. Typical assays to measure virulence of R. solanacearum in laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a novel inoculation protocol to analyze the replication of R. solanacearum upon infiltration into the leaves of Nicotiana benthamiana, in which gene expression has been altered using Agrobacterium tumefaciens. The protocol includes five major steps: 1) growth of N. benthamiana plants; 2) infiltration of A. tumefaciens; 3) R. solanacearum inoculation; 4) sample collection and bacterial quantitation; 5) data analysis and representation. The transient gene expression or gene silencing prior to R. solanacearum inoculation provides a straightforward way to perform genetic analysis of plant functions involved in the interaction between pathogen and host, using the appropriate combination of A. tumefaciens and R. solanacearum strains, with high sensitivity and accuracy provided by the quantitation of bacterial numbers in plant tissues.