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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.

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Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and ∼10 nm in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called 'Vetting & Analysis of RNA for in situ Hybridization probes' (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction.

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We developed public web sites and resources for data access, display, and analysis of plant small RNAs. These web sites are interconnected with related data types. The current generation of these informatics tools was developed for Illumina data, evolving over more than 15 years of improvements. Our online databases have customized web interfaces to uniquely handle and display RNA-derived data from diverse plant species, ranging from Arabidopsis (Arabidopsis thaliana) to wheat (Triticum spp.), including many crop and model species. The web interface displays the abundance and genomic context of data for small RNAs, parallel analysis of RNA ends/degradome reads, RNA sequencing, and even chromatin immunoprecipitation sequencing data; it also provides information about potentially novel transcripts (antisense transcripts, alternative splice isoforms, and regulatory intergenic transcripts). Numerous options are included for downloading data as tables or via web services. Interpretation of these data is facilitated by the inclusion of extensive repeat or transposon data in our genome viewer. We have developed graphical and analytical tools, including a new viewer and a query page for the analysis of phased small RNAs; these are particularly useful for understanding the complex small RNA pathways of plants. These public databases are accessible at https://mpss.danforthcenter.org.

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RNA interference (RNAi) is conserved in eukaryotic organisms, and it has been well studied in many animal and plant species and some fungal species, yet it is not well studied in fungal plant pathogens. In the rice blast fungus Magnaporthe oryzae, we examined small RNA (sRNA) and their biogenesis in the context of growth and pathogenicity. Through genetic and genomic analyses, we demonstrate that loss of a single gene encoding Dicer, RNA-dependent RNA polymerase, or Argonaute reduces sRNA levels. These three proteins are required for the biogenesis of sRNA-matching genome-wide regions (coding regions, repeats, and intergenic regions). The loss of one Argonaute reduced both sRNA and fungal virulence on barley leaves. Transcriptome analysis of multiple mutants revealed that sRNA play an important role in transcriptional regulation of repeats and intergenic regions in M. oryzae. Together, these data support that M. oryzae sRNA regulate developmental processes including, fungal growth and virulence.

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BACKGROUND: The rice blast fungus, Magnaporthe oryzae is a destructive pathogen of rice and other related crops, causing significant yield losses worldwide. Endogenous small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs) are critical components of gene regulation in many eukaryotic organisms. Recently several new species of sRNAs have been identified in fungi. This fact along with the availability of genome sequence makes M. oryzae a compelling target for sRNA profiling. We have examined sRNA species and their biosynthetic genes in M. oryzae, and the degree to which these elements regulate fungal stress responses. To this end, we have characterized sRNAs under different physiological stress conditions, which had not yet been examined in this fungus. RESULTS: The resulting libraries are composed of more than 37 million total genome matched reads mapping to intergenic regions, coding sequences, retrotransposons, inverted, tandem, and other repeated regions of the genome with more than half of the small RNAs arising from intergenic regions. The 24 nucleotide (nt) size class of sRNAs was predominant. A comparison to transcriptional data of M. oryzae undergoing the same physiological stresses indicates that sRNAs play a role in transcriptional regulation for a small subset of genes. Support for this idea comes from generation and characterization of mutants putatively involved in sRNAs biogenesis; our results indicate that the deletion of Dicer-like genes and an RNA-Dependent RNA Polymerase gene increases the transcriptional regulation of this subset of genes, including one involved in virulence. CONCLUSIONS: Various physiological stressors and in planta conditions alter the small RNA profile of the rice blast fungus. Characterization of sRNA biosynthetic mutants helps to clarify the role of sRNAs in transcriptional control.

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The analysis of cytosine methylation provides a new way to assess and describe epigenetic regulation at a whole-genome level in many eukaryotes. DNA methylation has a demonstrated role in the genome stability and protection, regulation of gene expression and many other aspects of genome function and maintenance. BS-seq is a relatively unbiased method for profiling the DNA methylation, with a resolution capable of measuring methylation at individual cytosines. Here we describe, as an example, a workflow to handle DNA methylation analysis, from BS-seq library preparation to the data visualization. We describe some applications for the analysis and interpretation of these data. Our laboratory provides public access to plant DNA methylation data via visualization tools available at our "Next-Gen Sequence" websites (http://mpss.udel.edu), along with small RNA, RNA-seq and other data types.

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