Current protocols最新文献

{"title":"Methods to Observe Plant Tissue Colonization by Fusarium oxysporum","authors":"Domingo Martínez-Soto,&nbsp;Gengtan Li,&nbsp;Li-Jun Ma","doi":"10.1002/cpz1.70152","DOIUrl":"https://doi.org/10.1002/cpz1.70152","url":null,"abstract":"<p><i>Fusarium oxysporum</i>, an important soil-borne pathogen, causes vascular wilts in more than 100 plant species, leading to billions of dollars in annual yield losses. Controlling <i>Fusarium</i> wilt diseases is challenging due to the persistence of pathogen spores in infested fields and the growing resistance to available fungicides. Understanding the molecular interactions between <i>F. oxysporum</i> and its host plants is crucial for developing novel control strategies, but studying these interactions is difficult because <i>F. oxysporum</i> invades plant roots long before wilt symptoms can be detected in above-ground tissues. To illuminate the hidden interactions between <i>F. oxysporum</i> and its plant hosts, we present three confocal microscopy protocols for visualizing fungal colonization in plant tissues and the associated plant responses. The first protocol employs wheat germ agglutinin–Alexa Fluor 488 and propidium iodide to stain fungal cells and plant host tissues, respectively. The second uses sirofluor to detect deposition of callose, a (1,3)-β-glucan polymer found in plant cell walls that plays a significant role in plant defense. The third utilizes fluorescent protein–tagged fungal isolates and a stable transgenic <i>Arabidopsis thaliana</i> line, providing a clean and easily accessible system for visualizing early infection stages. The protocols described here will shed light on underground plant-pathogen interactions, aiding researchers in unraveling the complex dynamics between diverse <i>F. oxysporum</i> pathotypes and their plant hosts.© 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Observation of <i>F. oxysporum</i> cells in the tomato stem vasculature</p><p><b>Basic Protocol 2</b>: Observation of callose deposition in <i>F. oxysporum</i>–colonized tomato plant roots</p><p><b>Basic Protocol 3</b>: Observation of fungal colonization in an <i>F. oxysporum</i>–<i>A. thaliana</i> model system</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244811","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":"From Genetic Association to Therapeutic Target: A Pipeline for Pleiotropic Gene Prioritization","authors":"Morgan Ewald,&nbsp;Erin Young,&nbsp;Michael Kuehn,&nbsp;Olivia Veatch","doi":"10.1002/cpz1.70148","DOIUrl":"https://doi.org/10.1002/cpz1.70148","url":null,"abstract":"<p>As our understanding of human health grows, we often see that similar biological dysfunction underlies the co-occurrence of various complex diseases. It remains difficult to determine if there are common genetic mechanisms contributing to clinically distinct conditions or if expression of both conditions relates to other shared risk factors. For example, in some situations, genetic variation may increase risk for one condition, and expression of this condition then increases risk for another disease. Identifying potentially pleiotropic genes is crucial for advancing the development of more effective treatment options, especially in instances where current therapies are insufficient. Genome-wide association studies (GWAS) provide cross-trait associations but do not provide the full functionality of how dysfunction in genes being tagged by GWAS hits are contributing to two or more distinct phenotypes. Fortunately, as other types of available data continue to grow exponentially (e.g., RNA-seq, mass spectrometry, mouse knock-out phenotype associations), these can be leveraged to help process GWAS results into meaningful information. The aim of this protocol is to provide clear instructions for using various databases and available software tools to identify key pleiotropic genes contributing to two distinct phenotypes of interest. The protocol uses information from various publicly available databases, including GWAS Catalog, Functional Mapping and Annotation (FUMA), Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT), International Mouse Phenotype Consortium (IMPC), STRINGdb, Pharos, and Cytoscape for network visualization. This pipeline, with code written in R and RStudio software, helps the user identify and generate hypotheses about shared genetic mechanisms contributing to their selected phenotypes of interest as well as prioritize genes of interest to functionally follow up in model systems that are more likely to be clinically relevant. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Pleiotropic gene prioritization pipeline for studies in model systems</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244596","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":"Quantitative Assessment of Mitochondrial Volumetric Transitions in Arabidopsis thaliana","authors":"Bryan Ocampo-Hernández,&nbsp;Delia Luisa Rodríguez-Bustos,&nbsp;Javier Plasencia,&nbsp;Manuel Gutiérrez-Aguilar","doi":"10.1002/cpz1.70156","DOIUrl":"https://doi.org/10.1002/cpz1.70156","url":null,"abstract":"<p>Mitochondria in plants typically appear as discrete spherical or slightly tubular organelles, with their morphology and volume serving as indicators of metabolic state and dysfunction. Measuring changes in mitochondrial volume is relatively straightforward in organisms lacking plastids. However, in chlorophyll-rich tissues, such assessments often require purification protocols that may compromise accuracy. Here, we present protocols for the quantitative assessment of mitochondrial volume transitions in leaf mesophyll cells of <i>Arabidopsis thaliana</i>. The methods are simple and highly sensitive and offer a reliable approach for studying mitochondrial morphology transitions under both physiological and stress conditions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Leaf mesophyll treatment and mitochondrial imaging</p><p><b>Basic Protocol 2</b>: Leaf mesophyll mitochondrial volume assessment</p><p><b>Basic Protocol 3</b>: Mitochondrial volume statistics</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70156","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144206343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Peptide–Oligonucleotide Conjugates: Catalytic Preparation in Aqueous Solution or On-Column","authors":"Marion Gras,&nbsp;Michael Smietana,&nbsp;Pauline Adler","doi":"10.1002/cpz1.70154","DOIUrl":"https://doi.org/10.1002/cpz1.70154","url":null,"abstract":"<p>Peptide–oligonucleotide conjugates (POCs) are synthesized through a novel catalytic and sustainable approach. The amide coupling between peptide and oligonucleotide is facilitated by 1,4-diazabicyclo[2.2.2]octane (DABCO) as catalyst and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) as a stoichiometric activating agent. Two distinct methods are described for the POC synthesis: the first involves an on-column conjugation in an anhydrous environment, while the second enables a post-synthetic conjugation in an aqueous buffer. This last approach has been successfully extended to an iterative process, offering significant potential for the development of DNA-encoded libraries.© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: On-column conjugation of an amino acid or of a peptide to an oligonucleotide</p><p><b>Support Protocol</b>: Deprotection of the Boc-protected amine of the lysine</p><p><b>Basic Protocol 2</b>: In-solution conjugation of an amino acid or of a peptide on an oligonucleotide</p><p><b>Basic Protocol 3</b>: Iterative process for the preparation of a peptide–oligonucleotide conjugate</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70154","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144197140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cover Image, Volume 5, Issue 5","authors":"","doi":"10.1002/cpz1.70159","DOIUrl":"https://doi.org/10.1002/cpz1.70159","url":null,"abstract":"<p>The cover image is based on the article Purification of recombinant histones and mononucleosome assembly by Jingjun Hong et al., https://doi.org/10.1002/cpz1.70155.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70159","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144140410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simple and Efficient Transformation and Gene Editing of Marchantia polymorpha Spores","authors":"Rebecca M. Yorker,&nbsp;Simon C. Deroles,&nbsp;Yanfei Zhou,&nbsp;Jennifer A. Tate,&nbsp;Kevin M. Davies,&nbsp;Nick W. Albert","doi":"10.1002/cpz1.70149","DOIUrl":"https://doi.org/10.1002/cpz1.70149","url":null,"abstract":"<p><i>Marchantia polymorpha</i> (Marchantia) has become a model species for liverwort studies, owing to its rapid growth <i>in vitro</i>, ease of propagation, simple genetics, small genome, haploid-dominant life cycle, and because it is amenable to genetic transformation. Efficient transformation provides a foundation for many molecular and genetic analyses. The protocol described here is a simple and robust procedure for transforming Marchantia for a variety of applications, including gene overexpression and CRISPR genome editing. This simplified <i>Agrobacterium tumefaciens</i>-mediated transformation protocol targets spores, using common <i>Agrobacterium</i> strains GV3101 or EHA105, and overcomes challenges experienced in other methods. Spores are sterilized and distributed over sterile filter papers, which effectively retain spores and regenerating spores (known as sporelings). This approach enables the sporelings to be transferred to different agar growth media at different stages of transformation. A critical feature is preculturing the spores with acetosyringone (AS) prior to co-cultivation with <i>Agrobacterium</i>. This treatment profoundly enhances the transformation rate, particularly for <i>Agrobacterium</i> strain GV3101. GV3101 is preferred for its rapid growth rate, simple transformation, and lack of a recombinase (<i>recA</i>), stabilizing plasmids. The protocol is suitable for transforming Marchantia with constructs for CRISPR gene editing. Editing efficiency can be increased by introducing a heat-shock treatment during the transformation procedure, which increases the proportion of plants with larger edited sectors, facilitating mutant identification and propagation. Constructs and strategies for both overexpression and multiplex genome editing with sgRNA arrays using new and existing vectors are described. Using this spore transformation protocol for CRISPR gene editing, we routinely achieve 60% to 70% mutation rates, significantly reducing the effort required to generate and isolate mutants for functional analyses. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: <i>Agrobacterium</i>-mediated transformation of <i>Marchantia polymorpha</i> spores</p><p><b>Support Protocol</b>: Induction of spores</p><p><b>APPENDIX 1</b>: Overexpression Vectors</p><p><b>APPENDIX 2</b>: Multiplex CRISPR using tRNA arrays</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70149","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144140418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of Conformation-Specific Monoclonal Antibodies for Integral Membrane Proteins","authors":"Natalie Sheldon,&nbsp;Gunasekaran Dhandapani,&nbsp;Junhoe Kim,&nbsp;Cathy J. Spangler,&nbsp;Chengli Fang,&nbsp;Jumi Park,&nbsp;Prashant Rao,&nbsp;Eric Gouaux","doi":"10.1002/cpz1.70142","DOIUrl":"https://doi.org/10.1002/cpz1.70142","url":null,"abstract":"<p>Antibodies and their antigen-binding fragments, including fragment antigen-binding domains (Fabs) and single-chain variable fragments (scFvs), are extraordinary tools in all fields of biology, particularly in neuroscience, where they have been utilized for imaging, detection, and quantification studies. Most antibodies bind to unstructured or linear epitopes. Conformation-specific antibodies, by contrast, bind to 3D epitopes, recognizing native conformations of the target antigen, and have proven highly useful in X-ray crystallography as crystallization chaperones and in cryo-electron microscopy as fiducial markers. Moreover, because conformation-specific antibodies recognize 3D shapes of the antigen, they often have exquisite specificity and are useful in immunofluorescence studies and in isolation of antigen from native tissues. Over the past decade, our group has devoted effort to developing murine monoclonal antibodies (mAbs) against important synaptic receptors, particularly ionotropic glutamate receptors (iGluRs) and their auxiliary proteins. We have developed reproducible methods for generating high-quality mAbs for structural, biochemical, and imaging studies. In this article, we show how to prepare proteoliposomes (PLs), carry out immunization and track the immune response, perform hybridoma generation, and analyze the specificity, cross-reactivity, and competition of mAb binding via enzyme-linked immunosorbent assay and fluorescence-detection size-exclusion chromatography. Our PL-based method produces high-affinity, conformation-specific antibodies targeting diverse synaptic membrane receptors in 4 months. Here, we describe the relevant protocols in detail and document the mAbs, Fabs, and scFvs that we have produced against iGluRs and their auxiliary subunits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Generation of conformation-specific antibodies for integral membrane proteins</p><p><b>Support Protocol 1</b>: Detection of conformational antibodies using ELISA</p><p><b>Basic Protocol 2</b>: Expression and purification of monoclonal antibodies and their derivatives</p><p><b>Support Protocol 2</b>: Concentration and clarification of insect cell supernatant for Fab purification</p><p><b>Support Protocol 3</b>: Measurement of ionotropic glutamate receptor binding kinetics using Octet BLI System</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70142","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144140489","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual Labeling of Newly Synthesized Proteins and Existing Proteins Using CG-SLENP","authors":"Bingbing X. Li,&nbsp;Xiangshu Xiao","doi":"10.1002/cpz1.70146","DOIUrl":"https://doi.org/10.1002/cpz1.70146","url":null,"abstract":"<p>This article describes a robust method for dual labeling of newly synthesized proteins and existing proteins. Assessing the properties of individual newly synthesized proteins from the bulk proteome is challenging due to difficulty in specifically isolating them. Previous methods such as non-natural amino acid labeling, isotope-labeled amino acid labeling, and puromycin labeling are effective for ensemble measurements. However, these existing methods do not support live-cell tracking or dynamic studies for a specific target protein. We designed a <span>c</span>hemical <span>g</span>enetics–based method for <span>s</span>elective <span>l</span>abeling of <span>e</span>xisting and <span>n</span>ewly synthesized <span>p</span>roteins (_CG-SLENP). Using nuclear lamin A (LA) tagged with a HaloTag (HaloTag-LA) as an exemplar protein and various Halo ligands, we describe _CG-SLENP for labeling existing proteins and newly synthesized proteins. This approach can label these proteins either one at a time or dually in the same live cell. This method holds great potential for broader applications to study any given protein of interest.©2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: _CG-SLENP labeling using clickable Halo ligands</p><p><b>Basic Protocol 2</b>: _CG-SLENP labeling using fluorescent Halo ligands</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144118061","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":"Purification of Recombinant Histones and Mononucleosome Assembly","authors":"Yiming Zhao,&nbsp;Jingjun Hong","doi":"10.1002/cpz1.70155","DOIUrl":"https://doi.org/10.1002/cpz1.70155","url":null,"abstract":"<p>The nucleosome is the basic unit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a <span>histone octamer</span>. Each histone octamer is composed of two copies each of the histone proteins <span>H2A</span>, <span>H2B</span>, <span>H3</span>, and <span>H4</span>. Nucleosomes play an important role in gene expression and regulation. While previous protocols use HPLC (high-performance liquid chromatography) to purify each histone and, after octamer reconstitution, subsequent FPLC (fast protein liquid chromatography) to purify the octamers. Here we present a method to carry out octamer reconstitution and mononucleosome assembly with FPLC only. This basic protocol describes a procedure for histone purification from <i>Escherichia coli (E. coli)</i>, their subsequent reconstitution as octamers, and assembly into mononucleosomes <i>in vitro</i>. Through this protocol, histone octamers and mononucleosomes can be reconstituted on a large scale without the use of HPLC. Overall, this protocol describes an effective new method for the reconstitution of octamers and assembly of mononucleosomes. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Purification of recombinant histones and mononucleosome assembly</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144118025","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":"Navigating the Scholarly Literature: A Practical Guide to Searching Effectively (Without Too Much Stress)","authors":"Kathryn P. Kohn","doi":"10.1002/cpz1.70138","DOIUrl":"https://doi.org/10.1002/cpz1.70138","url":null,"abstract":"<p>Effectively searching the scholarly literature is a fundamental academic skill. However, the process can be overwhelming due to the vast amount of available research and the complexity of academic databases. This overview article provides a practical guide to navigating the literature with confidence, outlining key strategies for identifying relevant sources, refining search queries, and troubleshooting common challenges. © 2025 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70138","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143930204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}