Methods in molecular biology最新文献

The yeast one-hybrid (Y1H) assay is a powerful molecular biology tool for studying protein-DNA interactions. This technique involves using a single DNA bait cloned upstream of a reporter gene in a yeast plasmid to determine whether a specific transcription factor (TF) or other DNA-binding protein (referred to as "prey") can bind to the DNA bait. Typically, this technique employs a complete cDNA library to obtain multiple prey proteins, which are then introduced into yeast cells containing the bait construct. In this protocol, we present a simplified method not centered on finding new prey from cDNA libraries but on analyzing specific target plant proteins. The protocol provides a step-by-step guide for cloning DNA-bait and protein-prey constructs, transforming yeast, and screening for reporter interactions.

Our protocol outlines a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay, which combines CRISPR/Cas12a technology with isothermal Recombinase Polymerase Amplification (RPA) for the rapid and specific detection of phytoplasma diseases in plants. This isothermal method utilizes RPA to amplify the target DNA fragment from the genomic DNA of phytoplasmas, followed by incubation with Cas12a nuclease and CRISPR RNAs (crRNAs) specifically designed to target unique phytoplasma DNA sequences. Upon initial cleavage of the amplified target DNA, Cas12a gains enzymatic activity to indiscriminately cleave single-stranded fluorescent oligonucleotide reporters, generating a fluorescent signal for highly sensitive detection of the pathogen. The protocol provides detailed instructions on: (i) sample collection and preparation; (ii) assay reaction setup, including RPA and Cas12a detection steps; (iii) reaction and detection conditions; and (iv) guidelines for accurately interpreting fluorescence data to detect phytoplasma DNA. This protocol is designed for researchers and agricultural professionals to effectively adopt and implement this advanced diagnostic technique.

Bioluminescence tagging has gained popularity as an effective tool for investigating infection processes of phytopathogenic bacteria. A critical consideration in employing this approach is to minimize the impact of the genetically introduced luciferase genes on bacterial fitness, while maximizing the intensity and stability of bioluminescence. Recently, the pBJ vector series was developed as all-in-one system for bioluminescence tagging in a wide range of Pseudomonadota, including the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pto). The pBJ vectors enable inducible transposition of the luxCDABE luciferase operon, derived from Photorhabdus luminescens, into a specific and neutral genomic location via Tn7 transposon. The resulting bioluminescent Pto strain, termed Pto-lux, emits stable and strong bioluminescence while maintaining bacterial fitness during plant infection. Moreover, bioluminescence-based assays using Pto-lux enable accurate quantification of in planta bacterial titers across diverse host plants, with a dynamic range of four orders of magnitude. In this chapter, we present detailed protocols for the bioluminescence-based bacterial growth assays in Arabidopsis thaliana and Marchantia polymorpha.

Transcriptomic profiling of plant-bacterial interactions provides critical insights into the molecular mechanisms underlying parasitism, commensalism, and mutualism. RNA sequencing (RNA-seq) enables the simultaneous analysis of plant and bacterial transcriptomes during colonization; however, integrated computational workflows specifically tailored for co-transcriptome analysis remain limited. Here, we present a step-by-step bioinformatics pipeline for analyzing co-transcriptome landscapes in plant-bacterial interactions. This workflow includes: (1) quality control and processing of raw RNA-seq data from both plant host and in-planta bacterial populations; (2) statistical analyses for differential gene expression; (3) prediction of orthologous bacterial genes and functional annotation of bacterial transcripts using the KEGG database; (4) integration and comparative analysis across multiple bacterial strains; and (5) correlation-based analysis of transcriptional dynamics between plants and bacteria. Designed for researchers with basic familiarity with command-line tools and R programming, this pipeline enables comprehensive analysis of plant-bacterial transcriptional interplay and facilitates hypothesis generation in both pathogenic and symbiotic contexts.

BN-PAGE (Blue Native-Polyacrylamide Gel Electrophoresis) is a non-denaturing electrophoretic technique used to analyze the molecular weight and oligomeric states of protein complexes under near-native conditions. NLRs (nucleotide-binding leucine-rich repeat proteins), which function as intracellular immune receptors in plants, form oligomeric higher-order complexes known as resistosomes upon activation by recognition of pathogen effectors-a mechanism elucidated through BN-PAGE and structural analyses. Here, we describe a method combining BN-PAGE with Agrobacterium-mediated complementation assay to investigate the resistosome formation of the NLR protein ZAR1 in Nicotiana benthamiana.

The histone H1 family comprises of essential components of chromatin, which bind to the linker DNA connecting individual nucleosomes and contribute to the formation of higher-order chromatin structures. Multiple histone H1 exist, each with distinct interactions with the nucleosome that specifically influence chromatin organization and nuclear functions. Histone H1 variants have been shown to play a role as drivers in cancer and may serve as biomarkers for patient stratification. To overcome the limitations associated with antibody- and RNA-based methods for analyzing histone H1, we developed a mass spectrometry (MS)-based label-free approach to simultaneously analyze all somatic histone H1 variants in patient-derived samples. Here, we describe how this method can be used for the analysis of low-amount clinical samples obtained through laser capture microdissection of tissue sections.

Subcellular fractionation of Euglena gracilis has been conducted for over 50 years in various forms by numerous research groups. The development of this technique is closely tied to the specific organelle or fraction required for specific purposes. In this chapter, we describe our approach to this process and discuss the insights we gain from it. Sucrose and iodixanol gradients are employed to separate the main organelles of interest; however, these methods alone do not lead to the complete purification of the organelles.

Post-transcriptional control is well established as a key mechanism of gene regulation in trypanosomatids. However, recent studies suggest that transcriptional regulation may also play a role, challenging long-standing dogma. The Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) provides a genome-wide overview of chromatin accessibility (i.e., whether chromatin is more open or closed), without requiring prior knowledge of chromatin markers which may be absent or poorly understood in trypanosomatids. Here, we present an optimized ATAC-seq protocol for use in Trypanosoma brucei, which has been used in both bloodstream and procyclic forms, and can be used to inform application of the method to other Euglenozoa. We also provide guidance on bioinformatic analysis, including integration of output files with established differential accessibility and RNA-seq data analysis pipelines.

Camelid single-domain antibodies (sdAbs), commercially known as Nanobodies™, possess remarkable properties that render them highly suitable as versatile tools for target discovery and product development. Interestingly, despite their successful and broad deployment in life sciences, sdAbs remain heavily underutilized in the field of molecular parasitology. In this chapter, we describe how we have employed an unbiased camelid immunization strategy to discover novel diagnostic biomarkers with sdAbs. This protocol shows the potential of camelid sdAbs as powerful tools for novel target discovery in kinetoplastid research.

Analysis of the cell cycle in kinetoplastid parasites involves the assessment of the replication of single copy organelles, such as the nucleus, kinetoplast, and flagellum, alongside the observation of cell cycle stage-associated morphological changes, e.g., cell shape changes and the appearance of a mitotic spindle or cytokinesis furrow, which together allow the cell cycle stage of individual parasites to be determined. To date, most kinetoplastid cell cycle analysis has been performed using light microscopy and/or flow cytometry of fixed cells, but while these methods have proven highly valuable, microscopy can be time-consuming and flow cytometry can lack resolution. We have previously shown that imaging flow cytometry offers significant benefits for depth and speed of analysis. This is due to its ability to directly link the high-throughput and quantitative nature of standard flow cytometry with the visual and spatial data of microscopy, over an extensive array of morphological and fluorescence parameters, which can be calculated for both brightfield and fluorescence images of each cell. Furthermore, the ability to automate image analysis ensures high throughput. Here, we provide a step-by-step guide to analyzing the cell cycle of live promastigote Leishmania mexicana using imaging flow cytometry. We outline a method for quantitative DNA staining in live L. mexicana promastigotes using Vybrant™ DyeCycle™ Orange and provide protocols, guidance, and example analysis templates for using an ImageStream^®X MkII imaging flow cytometer (Cytek) to acquire and analyze brightfield and fluorescence images of the parasite to determine cell cycle stage. We also detail how to employ mNeonGreen tagging of the orphan spindle kinesin, KINF, to provide greater resolution of cell cycle position. Our automated masking and gating pipeline enables rapid, high-throughput and semi-automated analysis of the L. mexicana cell cycle in live cells, in near real time, offering many advantages over conventional analysis methods. In addition, we envisage that this pipeline could be adapted to allow similar high-throughput analysis of the cell cycle of other kinetoplastid species and outline the approaches that could be taken to achieve this.