Methods in molecular biology最新文献

{"title":"End-Point PCR for Universal Detection of Phytoplasmas Based on the 16S rRNA Gene.","authors":"Bojan Duduk","doi":"10.1007/978-1-0716-5104-9_3","DOIUrl":"https://doi.org/10.1007/978-1-0716-5104-9_3","url":null,"abstract":"<p><p>End-point PCR, followed by restriction fragment length polymorphism (RFLP) or sequencing and sequence analysis of the 16S rRNA gene, is a cornerstone method for the universal detection and identification of phytoplasmas, including previously undescribed strains. This protocol describes the use of end-point PCR in direct, nested, or semi-nested systems, coupled with RFLP analysis using 17 restriction enzymes, to classify phytoplasmas into distinct 16Sr ribosomal groups and subgroups while addressing challenges such as inter-operon heterogeneity and mixed infections. Alternatively, sequencing of the PCR-amplified 16S rRNA gene, followed by sequence analysis (e.g., virtual RFLP or sequence homology), enables classification into either 16Sr groups/subgroups or 'Candidatus Phytoplasma' species. The method's sensitivity, cost-effectiveness, and compatibility with established classification frameworks make it invaluable for epidemiological studies, quarantine measures, and the delineation of 'Candidatus Phytoplasma' species. By providing a clear framework for the precise diagnosis of phytoplasma-associated diseases in diverse plant and insect hosts, this protocol supports rapid responses to outbreaks and helps mitigate the economic impact of phytoplasmas.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3008 ","pages":"21-35"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145959711","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Imp Gene Analysis for Diagnosis and Genotyping of Flavescence Dorée Related Phytoplasmas.","authors":"Luisa Filippin, Graziana Da Rold, Elisa Angelini","doi":"10.1007/978-1-0716-5104-9_4","DOIUrl":"https://doi.org/10.1007/978-1-0716-5104-9_4","url":null,"abstract":"<p><p>Imp is a gene coding for the immunodominant membrane protein Imp that is supposed to be involved in host-pathogen interactions. It was identified in some 'Candidatus Phytoplasma' species and showed a considerable sequence variability. For this characteristic it can be exploited to complement conventional phytoplasma classification based on the conserved 16S rRNA gene, enhancing the differentiation of closely related phytoplasma strains. This protocol describes a method for amplification and sequence analysis of the imp gene of Flavescence dorée (FD) related phytoplasmas, that can be useful for detecting and genotyping the strains involved, for example, in the complex FD epidemiology.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3008 ","pages":"37-50"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145959730","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cleavage Under Targets and Tagmentation (CUT&Tag) Protocol for Plants.","authors":"Xiao-Yuan Tao, Jia-Wei Wang","doi":"10.1007/978-1-0716-4972-5_3","DOIUrl":"https://doi.org/10.1007/978-1-0716-4972-5_3","url":null,"abstract":"<p><p>CUT&Tag (Cleavage Under Targets and Tagmentation) is a powerful method for chromatin profiling based on an enzyme-tethering strategy. Compared with chromatin immunoprecipitation (ChIP) assay, CUT&Tag requires smaller amount of cell/nuclei input and generates data with higher signal-to-noise ratio, which has been widely applied in profiling histone modifications and protein-DNA interactions. Here, we describe a detailed protocol for bulk-cell CUT&Tag in plants and briefly introduced other derivative CUT&Tag methods.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2985 ","pages":"37-50"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145948902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual-Luciferase Assay System for the Quantification of Promoter Activity.","authors":"Patricia Ballester, Cristina Ferrándiz","doi":"10.1007/978-1-0716-4972-5_5","DOIUrl":"https://doi.org/10.1007/978-1-0716-4972-5_5","url":null,"abstract":"<p><p>Transient luciferase reporter assays are a widely used and powerful tool for investigating promoter activity and gene regulation in plant systems. These assays make use of the enzymatic conversion of a luciferin substrate into bioluminescence to provide a highly sensitive and reproducible measure of transcriptional activity. The dual-luciferase system, incorporating Firefly luciferase (LUC) as the primary reporter and Renilla luciferase (REN) as an internal control, enhances experimental accuracy by normalizing for variations in infiltration efficiency, tissue viability, and environmental conditions. Agroinfiltration-mediated transient expression in Nicotiana benthamiana leaves enables rapid and high-throughput analysis of transcriptional regulation, including the functional characterization of transcription factors (TFs), cis-regulatory elements, and enhancer/silencer activity. This approach facilitates systematic promoter dissection through site-directed mutagenesis, deletion mapping, and combinatorial TF studies to uncover complex regulatory interactions. Here, we present a detailed protocol for performing dual-luciferase transient expression assays, emphasizing best practices for ensuring robust and reproducible results.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2985 ","pages":"65-74"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145948905","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"TurboID-Based Proximity Labeling in Plants.","authors":"Chaonan Shi, Huang Tan, Rosa Lozano-Durán","doi":"10.1007/978-1-0716-4972-5_10","DOIUrl":"https://doi.org/10.1007/978-1-0716-4972-5_10","url":null,"abstract":"<p><p>Proximity labeling (PL) techniques have emerged as powerful approaches for the study of protein associations in vivo. In this chapter, we present a step-by-step TurboID-based PL protocol to define the \"proxiome\" of a protein of interest in plants, from the generation of the fusion protein to the analysis of the results, using the publicly available Ti-TAN plasmid collection.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2985 ","pages":"133-144"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145949044","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Standardized Protocols and Bioinformatic Pipelines for Conducting DAP-seq Experiments in Non-model Plant Crops.","authors":"Chen Zhang, Chiara Foresti, Luis Orduña, Miaomiao Li, Gabrielle Magon, Meng-Bo Tian, Alvaro Vidal, Antonio Santiago, David Navarro-Payá, Iñigo Agirre, Alessandro Vannozzi, Sara Zenoni, Shao-Shan Carol Huang, José Tomás Matus","doi":"10.1007/978-1-0716-4972-5_1","DOIUrl":"https://doi.org/10.1007/978-1-0716-4972-5_1","url":null,"abstract":"<p><p>The cistrome comprises genomic loci that regulate gene expression, playing a crucial role in defining cellular identity and function. Analyzing cistrome data reveals key molecular mechanisms underlying grapevine growth, development, and environmental responses. Identifying transcription factors that bind specific DNA sequences allows researchers to dissect the complex regulatory networks controlling gene expression. Moreover, this analysis can help pinpoint targets for crop improvement, as traits like fruit quality, disease resistance, and abiotic stress tolerance are often regulated by transcription factors. DNA affinity purification sequencing (DAP-seq) is a high-throughput, cost-effective method for mapping the cistrome, providing valuable insights into transcriptional regulation. This technique relies on the in vitro affinity purification of genomic DNA-protein complexes, followed by high-throughput sequencing of eluted DNA fragments. Unlike other in vitro DNA-binding assays, such as protein-binding microarrays (PBM) and systematic evolution of ligands by exponential enrichment (SELEX), DAP-seq allows transcription factors to interact directly with plant-derived genomic DNA, capturing all potential binding sites. The resulting data closely resemble those from chromatin immunoprecipitation sequencing (ChIP-seq) but are obtained much faster. Initially developed in Arabidopsis, DAP-seq has since been applied to several crops, including maize, tomato, and grapevine, generating extensive cistrome datasets and deepening our understanding of gene regulatory regions. However, despite its power in elucidating crop biology, DAP-seq faces certain limitations, particularly concerning the size and complexity of plant genomes. This chapter presents detailed protocols for DAP-seq studies aimed at the unbiased identification of transcription factor binding sites in crops. Additionally, we outline a standardized pipeline for DAP-seq data analysis, encompassing raw sequencing data processing (i.e., trimming, filtering, and read alignment), as well as peak calling and motif discovery analysis. This approach enables the efficient and scalable identification of transcription factor binding profiles in diverse crop species.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2985 ","pages":"3-23"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145949054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Yeast One-Hybrid (Y1H) Assay for Single Promoter-Transcription Factor Interaction in Higher Plants.","authors":"Aileen Turner, Paz E Zuñiga, Carlos R Figueroa","doi":"10.1007/978-1-0716-4972-5_6","DOIUrl":"https://doi.org/10.1007/978-1-0716-4972-5_6","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2985 ","pages":"75-87"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145949065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid and Sensitive Detection of Phytoplasma Diseases Using a CRISPR/Cas12a DETECTR Assay Combined with Isothermal Recombinase Polymerase Amplification.","authors":"Wei Wei, Yinong Yang, Justin Shih","doi":"10.1007/978-1-0716-5104-9_6","DOIUrl":"https://doi.org/10.1007/978-1-0716-5104-9_6","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3008 ","pages":"63-74"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145958725","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bioluminescence-Based Growth Quantification of the Phytopathogenic Bacterium Pseudomonas syringae pv. tomato DC3000.","authors":"Yuga Fujinawa, Yijia Yan, Hirofumi Nakagami, Akira Mine","doi":"10.1007/978-1-0716-5138-4_16","DOIUrl":"https://doi.org/10.1007/978-1-0716-5138-4_16","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3012 ","pages":"241-251"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146125824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bioinformatics Workflow for Co-Transcriptome Analysis of Plant-Bacterial Interactions.","authors":"Ying Tang, Kenichi Tsuda","doi":"10.1007/978-1-0716-5138-4_13","DOIUrl":"https://doi.org/10.1007/978-1-0716-5138-4_13","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3012 ","pages":"181-223"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146125832","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}