Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros
{"title":"Inoculation of Maize Roots with <i>Globisporangium ultimum</i> var. <i>ultimum</i> to Study Pythium Root Rot.","authors":"Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros","doi":"10.1101/pdb.prot108640","DOIUrl":"10.1101/pdb.prot108640","url":null,"abstract":"<p><p>Maize is a globally important field crop for food and fuel production. Yield can be affected early in the growing season by oomycete and fungal pathogens that cause root rot or prevent seed germination. The diseases caused by these pathogens are referred to as seedling blights, root rots, or damping off. Pythium root rot is one of the most significant of these diseases. The disease is caused by multiple species of the oomycete genera <i>Globisporangium</i> and <i>Pythium</i> and results in significant yield losses due to reduced seed germination and reduced vigor of surviving seedlings. In this protocol, we mimic the natural infection process by mixing the inoculum into the potting media in which seeds are planted. Then, we flood the seeds daily for several days in large plastic totes to induce flooding conditions. Disease severity is assessed using stand counts and measuring root mass and length. This protocol allows researchers to investigate quantitative differences in disease symptoms, isolate aggressiveness, as well as levels of host resistance. This protocol was developed for the pathogen <i>Globisporangium ultimum</i> var. <i>ultimum</i>, but it can be adapted for other species.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215157","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}
Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros
{"title":"Corrigendum: Inoculation of Maize Roots with <i>Globisporangium ultimum</i> var. <i>ultimum</i> to Study Pythium Root Rot.","authors":"Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros","doi":"10.1101/pdb.Corr108654","DOIUrl":"https://doi.org/10.1101/pdb.Corr108654","url":null,"abstract":"","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144648775","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":"The <i>Drosophila</i> Larval Neuromuscular Junction: Developmental Overview.","authors":"James Ashley, Robert A Carrillo","doi":"10.1101/pdb.top108449","DOIUrl":"10.1101/pdb.top108449","url":null,"abstract":"<p><p>For decades, the <i>Drosophila</i> larval neuromuscular junction (NMJ) has been a go-to model for synaptic development. This simple, accessible system is composed of a repeating pattern of 33 distinct neurons that stereotypically innervate 30 muscles. Fundamental mechanisms that underlie diverse aspects of axon pathfinding, synaptic form, and function have been uncovered at the NMJ, and new pathways continue to be uncovered. These discoveries are fueled by the ease of dissections and an extensive array of techniques. Chief among these techniques are various microscopy approaches, including super-resolution and electron microscopy. Functionally, the <i>Drosophila</i> NMJ is glutamatergic, similar to the vertebrate central synapses, making it a great model to study normal development and neurological diseases. Here we provide a brief overview of the larval neuromuscular system, highlighting the connectivity patterns, development, and some of the mechanisms underlying these processes.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108449"},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310237","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":"Immunohistochemistry and Morphometric Analysis of <i>Drosophila</i> Larval Body Wall Neuromuscular Junction Preparations.","authors":"James Ashley, Robert A Carrillo","doi":"10.1101/pdb.prot108500","DOIUrl":"10.1101/pdb.prot108500","url":null,"abstract":"<p><p>The <i>Drosophila</i> neuromuscular junction (NMJ) is an excellent model for studying vertebrate glutamatergic synapses. Researchers have uncovered fundamental mechanisms at the fly NMJ that are conserved in higher-order organisms. To gain molecular and structural insight into these and other structures, immunolabeling is invaluable. In this protocol, we describe how to use immunolabeling to visualize embryonic/larval presynaptic and postsynaptic structures at the NMJ. We also include details about amplification of weak immunohistochemistry signals and how to use these signals to quantify synaptic growth via bouton counting. Boutons are bead-like structures at motor axon terminals that house synapses, and the number of boutons reflects the size of the NMJ. We also describe how to identify the different bouton types.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108500"},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310235","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":"Using the Proximity Ligation Assay to Visualize Colocalization of Proteins at the <i>Drosophila</i> Larval Neuromuscular Junction.","authors":"James Ashley, Robert A Carrillo","doi":"10.1101/pdb.prot108502","DOIUrl":"10.1101/pdb.prot108502","url":null,"abstract":"<p><p>In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized. Early work relied on fluorescent signals to determine whether proteins localized to the pre- and postsynaptic regions. As more synaptic molecules were identified, determining the localization of these proteins relative to each other became important. Conventional microscopy lacks the resolving power to assess whether two proteins are within an appropriate distance to bind directly or be part of a larger complex. Super-resolution and immunoelectron microscopies can improve spatial resolution, but these techniques can be difficult to execute and troubleshoot, and access to these instruments is limiting. However, another approach, proximity labeling, overcomes many of these limitations by using a DNA secondary label that can only be amplified if the two proteins of interest are within 40 nm of each other, which is ∼5× greater than the resolving power of conventional microscopy. In this protocol, we describe the use of the proximity ligation assay, which combines immunohistochemistry with DNA amplification, to reveal protein colocalization in the <i>Drosophila</i> NMJ.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108502"},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310238","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":"<i>Drosophila</i> Late Embryonic through Late Larval Stage Body Wall Dissection: Dissection Tools and Techniques.","authors":"James Ashley, Robert A Carrillo","doi":"10.1101/pdb.prot108499","DOIUrl":"10.1101/pdb.prot108499","url":null,"abstract":"<p><p>One of the challenges of studying synaptic structure and function is accessibility. Some of the earliest readily identifiable and accessible synapses were from the frog and various arthropods. To address questions regarding mechanisms that underlie synaptic development and function, genetically tractable systems were required, and researchers turned to the <i>Drosophila melanogaster</i> embryonic/larval neuromuscular preparation. <i>Drosophila</i> embryos are transparent and can be labeled with antibodies or probes and imaged in whole-mount preparation for structural analysis. Embryos can also be dissected to visualize the entire body wall musculature as well as finer details including live protein trafficking and protein-protein interactions. Whereas younger dissected embryos can be mounted directly onto charged slides, more mature embryos and larvae develop a cuticle that impedes this adherence, so different techniques must be applied. In this protocol, we detail how to manufacture dissection tools and collect embryos, and discuss the individual steps of dissecting late-stage embryos, early first-instar larvae, and late-stage third-instar larvae.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108499"},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310233","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":"Cell Ablation Techniques for the Larval <i>Drosophila</i> Neuromuscular System.","authors":"James Ashley, Robert A Carrillo","doi":"10.1101/pdb.prot108503","DOIUrl":"10.1101/pdb.prot108503","url":null,"abstract":"<p><p>Tissue development requires local and long-distance communication between cells. Cell ablation experiments have provided critical insights into the functions of specific cell types and the tissue surrounding the dead cells. In the <i>Drosophila</i> neuromuscular system, ablation of motor neurons and muscles has revealed the roles of the ablated cells in axon pathfinding and circuit wiring. For example, when muscles are denervated due to laser ablation of their motor neuron inputs, they receive ectopic innervation from neighboring motor neurons. Here, we describe two methods of specific cell ablation. The first is a genetic ablation approach that uses <i>GAL4</i> (ideally expressed in a small subset of cells) to drive expression of cell death genes <i>reaper</i> and <i>head involution defective</i> The second method relies on reactive oxygen species produced by light activation of the <i>Arabidopsis-</i>derived Singlet Oxygen Generator, miniSOG2, expressed in a subset of cells. For the latter, the precision stems from both the <i>GAL4</i> and the restricting of the blue-light stimulation area.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108503"},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310234","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":"Labeling of Cell Surface Proteins at the <i>Drosophila</i> Larval Neuromuscular Junction Using Binding Partner Peptides.","authors":"James Ashley, Robert A Carrillo","doi":"10.1101/pdb.prot108501","DOIUrl":"10.1101/pdb.prot108501","url":null,"abstract":"<p><p>Determining the precise localization of interacting proteins provides fundamental insight into their putative function. Classically, immunolabeling of endogenous proteins or generating tagged versions of proteins has been used to localize interacting proteins. However, in many cases, the interacting partner of a protein of interest is unknown. For cell surface proteins, it is possible to determine the localization of interacting proteins if one of the binding partners is known. This approach is based on generating purified, recombinant, tagged extracellular domains (ECDs) of a protein of interest, and incubating tissue to allow the recombinant protein to bind to its interacting partner(s). In this protocol, we detail the cloning of secreted, tagged ECDs from cell surface proteins, transfection of cloned plasmids into S2 cells, collection of secreted domains, concentration of the cell culture medium to enrich for the ECDs, and labeling of tissue with these ECDs.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108501"},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310236","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}
Miriam Nancy Salazar-Vidal, Melissa A Draves, Sarah L Fitzsimmons, Zachary B Traylor, Sherry Flint-Garcia
{"title":"How to Harvest and Store Corn (<i>Zea mays</i>).","authors":"Miriam Nancy Salazar-Vidal, Melissa A Draves, Sarah L Fitzsimmons, Zachary B Traylor, Sherry Flint-Garcia","doi":"10.1101/pdb.prot108639","DOIUrl":"https://doi.org/10.1101/pdb.prot108639","url":null,"abstract":"<p><p>Harvest scheduling, seed drying, and good storage practices are essential for maize research to avoid negative impacts on the quality of the seeds and to maximize seed viability. Embryo growth and accumulation of energy reserves in the endosperm are completed ∼40 days after pollination, of which the last 10-20 days are devoted to maturation and desiccation. Seed maturity is affected by many factors including temperature, day length, humidity, and soil moisture. Once seeds are mature, they must be harvested. Hand harvesting, which allows for greater control and minimizes ear damage, is primarily used in genetic nurseries and general research because each genotype is represented by a small number of plants. Hand harvesting is also used where there is a mix of manually pollinated and undesired open pollinated ears, and can be a selective harvest depending on the research objectives. However, hand harvesting is more labor-intensive and time-consuming compared to combine harvesting. If harvesting a yield trial, where the purpose is to collect yield data and identify promising genotypes, the use of a combine (not described in this protocol) is critical to consistently capture grain weight, moisture, and test weight for each plot. Following harvest, materials must be dried to the appropriate moisture content before storage. Corn is typically stored using the \"active collection\" model, with temperatures set between 5°C and 10°C and low relative humidity. This harvest protocol is intended to assist laboratories that are new to maize research and may be modified based on project goals, genetic material, equipment, or available space.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215152","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}