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Infection-associated chronic illnesses (IACIs) encompass a spectrum of poorly understood syndromes often marked by significant neurologic and multisystem symptoms following an infectious event. This review focuses on several diseases representative of the IACI spectrum. These are post-treatment Lyme disease syndrome (PTLDS), long COVID, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and multiple sclerosis (MS). Their clinical and biological complexity, combined with a lack of clear diagnostic criteria and objective available laboratory biomarkers, makes them difficult to distinguish from conditions with overlapping features. This presents challenges for research studies, as well as diagnosis and clinical management. This diagnostic ambiguity, coupled with heterogeneous patient presentations, has led to challenges in research, including misclassification of study participants and inconsistent or irreproducible findings. Some PTLDS research exemplifies these issues, which also extend to other IACIs. To advance the field, we highlight key methodological refinements and approaches for studying IACIs, including rigorous participant selection, standardized sample collection protocols, and the use of appropriate control groups, including those with microbiologic proof of the initial infection when known and technologically feasible. We also address broader influences on research quality, such as stigma, historical neglect, and the urgency to find treatments, which have contributed to the proliferation of poorly controlled studies and questionable practices. Drawing lessons from past challenges, we propose a path forward grounded in fit-for-purpose methodological rigour to improve scientific understanding and support evidence-based therapeutic development for IACIs.

Phage display technology is enabled by genetic fusion of a foreign protein domain to a phage coat protein, without interfering with the phage's ability to replicate by infecting bacterial host cells. The displayed domain is exposed on the phage particle (virion) surface, where it can interact with molecules or other substances in the surrounding medium; in this regard, it acts like a normal protein. However, it possesses a superpower that is unavailable to ordinary proteins: It is easily replicated in great abundance because it is attached to a replicating virion whose genome includes its coding sequence. The main way this technology is exploited is construction of huge phage display "libraries," comprising billions of phage clones, each displaying a different protein domain, and each represented by thousands, millions, or billions of genetically identical virions-all mixed together in a single vessel. Surface display allows exceedingly rare virions whose displayed protein domains happen to bind a user-defined molecule or other substance-generically called the "selector"-to be isolated from such libraries by an affinity selection process. The yield of selector-binding virions is much too low to be of practical use, but their number is readily increased by many orders of magnitude by propagating the virions in host bacteria in culture. This overview is a critical review of recent developments of this technology. It does not review the entire arena of contemporary phage display; there is special emphasis on phage display's most prominent application, phage antibodies, in which the displayed domain is an antibody domain, and the selector is an antigen of interest.

Properly characterizing the stages of corn growth is critical to conducting successful experiments in maize genetics and breeding. Specifically, accurately identifying stages of growth is required to perform developmentally dependent sampling or data collection, to predict time to flowering and seed maturation, and to allow for comparisons between different lines and populations based on developmental time. In this protocol, we summarize previous knowledge about maize development and describe how to monitor these stages in the reference inbred line B73, a yellow dent corn.

The human immune system evolved to defend against the panoply of microbial threats. By harnessing such ability, vaccines have cumulatively saved hundreds of millions of lives. Despite such tremendous success, there have also been remarkable failures, such as the lack of a clinically proven vaccine against Staphylococcus aureus (SA), which continues to pose an urgent public health threat. In practice, it has proven challenging to identify the molecular basis for relevant epitopes for this pathogen. Here, we summarize our experience implementing an integrated approach using phage display technology for the identification of B-cell epitopes of microbial virulence factors, which we developed with a focus on SA. This approach was used to define minimal B-cell epitopes of the staphylococcal leucocidin family of pore-forming toxins (PFTs) that have been implicated in staphylococcal clinical infection. Our methodology provides proof of principle for an approach well suited for the rapid and efficient generation of modular protein-based vaccines for protection from clinical infection, which can be used to target pathogens for which no vaccine is currently available.

Genetic engineering techniques are essential for both plant science and agricultural biotechnology, enabling functional genomics studies, dissection of complex traits, and targeted crop improvement. Among the various genetic tools currently in use, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based genome editing has emerged as a transformative technology due to its precision, versatility, and ease of use. In particular, CRISPR-Cas9 has become the most widely adopted platform for genome manipulation in plant systems, including maize, owing to its high editing efficiency, multiplexing capabilities, and scalability for diverse applications. This review highlights the biological significance and technical considerations necessary to implement CRISPR-Cas9 in maize. We discuss critical components for successful editing, including the selection of strong and tissue-appropriate promoters for Cas gene and guide RNA expression, codon optimization of Cas nuclease genes, effective guide RNA design, and multiplexing strategies using RNA polymerase III (Pol III)- or Pol II-dependent promoter-driven polycistronic expression systems. Additionally, we provide insights into vector construction methodologies and reliable genotyping techniques to detect and validate genome edits. Together, these elements constitute a practical framework for deploying genome editing in maize research and breeding. By optimizing these parameters, researchers can enhance the efficiency and accuracy of CRISPR-mediated genome modifications, accelerating functional genomic discovery and the development of improved maize varieties tailored to meet future agricultural demands.

Maize significantly contributes to food and fuel production. Yields can be reduced due to foliar diseases, which reduce photosynthetic leaf area. The bacterial foliar disease Goss's wilt (caused by Clavibacter nebraskensis) can cause significant yield losses in susceptible maize varieties. C. nebraskensis can infect leaves through wounds and colonize the vascular tissue of the leaf. We present a protocol that replicates this process with the use of a "clapper" with pins on one end to create wounds and a sponge soaked in inoculum on the other end, which allows for efficient field inoculations of maize leaves. Disease severity is then rated on a percentage scale multiple times over the season to generate an area under disease progress curve (AUDPC). Genetic host resistance is one of the most effective forms of foliar disease control in maize, as there are few effective forms of chemical control for bacterial diseases that affect maize. Screening for resistance in diverse germplasm, or for fine mapping a specific resistance gene, requires inoculating large populations in the field for obtaining phenotypic data. Our high-throughput protocol allows for large-scale disease evaluations and is useful for finding forms of genetic resistance or to understand plant-pathogen interactions of bacterial foliar pathogens.

Plant embryogenesis encompasses the biological processes wherein the zygote (fertilized egg) undergoes cell division, cell expansion, and cell differentiation to develop histological tissue layers, meristems, and various organs comprising the primordial body plan of the organism. Studies of embryogenesis in the agronomically important maize crop advance our understanding of the fundamental mechanism of plant development, which, upon translation, may advance agronomic improvement, optimization of conditions for somatic embryogenesis, and plant synthetic biology. Maize embryo development is coordinated temporally and spatially and is regulated by interactive genetic networks. Single-cell RNA sequencing (RNA-seq) and spatial transcriptomics are powerful tools to examine gene expression patterns and regulatory networks at single-cell resolution and in a spatial context, respectively. Single-cell technology enables profiling of three-dimensional samples with high cellular resolution, but it can be difficult to identify specific cell clusters due to a lack of known markers in most plant species. In contrast, spatial transcriptomics provide transcriptomic profiling of discrete regions within a sectioned, two-dimensional sample, although single-cell resolution is typically not obtained and fewer transcripts per cell are detected than in single-cell RNA-seq. In this review, we describe the combined use of these two transcriptomic strategies to study maize embryogenesis with synergistic results.

Understanding how the auxin hormone signaling pathway components come together to orchestrate cellular responses is key to engineering the growth and development of maize. Although a variety of techniques exist to measure auxin activities in plants, many are time- and resource-intensive or do not easily allow for high-throughput quantitative measurement of component libraries. The AuxInYeast system is a synthetic biology tool that facilitates complex biochemical analysis of the auxin hormone signaling pathway from essentially any plant. AuxInYeast uses Saccharomyces cerevisiae yeast as a heterologous expression platform for auxin signaling pathway components with fluorescent tags that facilitate measurement of auxin perception, repression, and activation. This protocol describes how to use fluorescence flow cytometry for these AuxInYeast experiments. As a case study, we focus on AuxInYeast strains built to measure maize auxin perception (i.e., those that express receptors and fluorescently tagged repressors that degrade upon auxin exposure). This protocol describes two different types of cytometry assays. The Steady-State Assay measures the extent of auxin-induced repressor degradation at one or two time points across many AuxInYeast strains and is particularly useful for initial assessment of whether auxin-induced degradation occurs and for dose response assays. The Time-Course Assay is used to measure auxin-induced repressor degradation dynamics over 2-3 h in a smaller number of strains. It is most useful for assessing the range of degradation rates across sets of repressors or receptors, and to precisely determine the impact of mutations and natural variation on degradation rate.

Maize is a globally important grain crop that is important for food and fuel. Northern corn leaf blight, caused by Exserohilum turcicum, is an important fungal foliar disease of maize that is highly prevalent and causes yield losses globally. Microscopy can be used to visualize plant-fungal interactions on a cellular level, which enables pathology and genetics studies. Host resistance and isolate aggressiveness can be characterized at different stages of disease development, which enables a more detailed understanding of the pathogenesis process and host-pathogen interactions. Our protocol outlines an efficient, cost-effective method for staining E. turcicum tissue on inoculated maize leaves and visualizing samples using a compound fluorescence microscope. This protocol uses KOH treatment followed by aniline blue staining, which stains glucans present in plant and fungal cell walls, and samples are visualized using fluorescence microscopy. Quantitative data about fungal structures including the conidia, hyphal structures, and appressoria, the structures formed to push through the plant leaf surface after conidia have germinated, can be obtained from the images generated using this technique. Visualization of these structures can help pathologists understand plant-pathogen interactions for maize and E. turcicum This method has advantages over other methods because the stain is less toxic than other available stains, samples can be processed in a more high-throughput manner than other protocols, and the required supplies are relatively inexpensive.

Seed mutagenesis using alkylating chemical agents such as ethyl methanesulfonate (EMS) can generate somatic and germinal mutations in many plant species. In monoecious plants like maize, the sperm- and egg-producing reproductive germlines are derived from distinct cell lineages in the embryo. This separation results in independent mutations inherited via the egg and sperm lineages and prevents the recovery of recessive mutant phenotypes in diploid progeny after the first round of self-pollination. Thus, two generations of self-pollination are required to screen for recessive mutations when conducting seed mutagenesis. The additional time and manual self-pollination make this approach laborious. However, a high mutation rate and the ability to screen for somatic sectors in heterozygous mutant plants and other defined genetic backgrounds make seed mutagenesis an effective but underutilized mutagenesis tool for maize research. This protocol provides the directions and optimization steps to perform effective seed mutagenesis in maize. A high frequency of somatic mutations from seed mutagenesis can be achieved, but comes at the expense of poor and disordered growth, failure to form reproductive structures, and low or no seed production at high EMS concentrations or long contact times. In experiments where germinal mutations are a goal, an optimum dose of EMS is required in the first generation. Maize genetic backgrounds vary in their sensitivity to EMS, requiring some pilot testing in new genetic backgrounds. Researchers using this protocol can carry out seed mutagenesis safely and effectively to develop libraries of mutants or alleles for various experiments.

Creating mutations in maize has provided key foundational information for our mechanistic understanding of genetics, evolution, and even the role of chromosomes as units of inheritance. Chemical mutagenesis is used in biological research to create novel genetic variation. Ethyl methanesulfonate (EMS) is an alkylating agent and a highly potent and frequently used mutagen. EMS mutagenesis can be used to identify genes based on phenotypes induced by mutagenesis (forward genetics) and to validate the functions of genes by independently creating multiple mutant alleles in known genes (reverse genetics). Due to our ability to collect huge quantities of maize pollen and to easily apply pollen to the silks of maize ears to conduct pollination and achieve hundreds of fertilization events, pollen EMS mutagenesis is uniquely facile in maize. While pollen EMS mutagenesis is commonly performed, treatment of maize seeds with EMS is also highly effective, and can be used for certain research objectives that are difficult to achieve with pollen mutagenesis, such as recovering mutant sectors. The alkylation of guanine residues by EMS primarily results in G > A or C > T transitions in the DNA, making the molecular profiling of mutations caused by EMS easy, with an extremely low false positive rate. EMS is hydrophilic, has a moderate half-life in water, and is sensitive to light and high temperatures. With appropriate precautions in research settings, EMS can be relatively safe to handle. Here, we provide an introduction to chemical mutagenesis via EMS, including some history on its use in maize and the considerations for the effective and safe design of mutagenesis experiments with EMS in maize.

Synthetic biology approaches merge the tenets of engineering with established biological techniques to answer fundamental questions about living systems and to engineer biological forms and functions. Following the engineering principle of design-build-test-iterate, this review serves as a guide to applying synthetic principles and approaches in maize. We outline strategies for (1) choosing the optimal model organism to serve as a heterologous chassis for maize signaling pathways, (2) designing and building biological parts and devices to express pathway components, (3) choosing an analytical technique to measure pathway function, and (4) optimizing and troubleshooting the designed system. Auxin is a hormone that is essential for plant growth and development, regulating cellular proliferation and differentiation. Considering the importance of auxin for maize development in aerial and underground tissue, it was an obvious starting point for synthetic biology approaches. We use the maize nuclear auxin response recapitulated in yeast (AuxInYeast) system to showcase the power of heterologous expression approaches for testing fundamental attributes of the evolution, genetics, and biochemistry of signaling pathways that may be challenging to assay in planta. This approach involves co-expression of maize auxin signaling components in Saccharomyces cerevisiae coupled with fluorescence flow cytometry to quantify signaling activity. We and others have used this system to interrogate the dynamics of pathway signaling, interactions between paralogous components, and the adaptation of auxin signaling over large evolutionary distances. Thus, the AuxInYeast system is a fast, high-throughput, hypothesis-generating platform that can be readily adapted by the maize community to creatively answer questions about fundamental maize biology and to drive development of novel tools for breeding and plant engineering.

Maize is an important food and fuel crop globally. Ear rots, caused by fungal pathogens, are some of the most detrimental maize diseases, due to reduced grain yield and the production of harmful mycotoxins. Mycotoxins are naturally occurring toxins produced by certain fungal species that can cause acute and chronic health issues in humans and animals that consume mycotoxin-contaminated grain. Pathogens can infect the developing ear through silks, or through wounds in the ears produced by pests. Plants naturally develop genetic resistance to pathogens. The maize genes involved in resistance to the pathogen may be different, depending on whether the ear was infected via silks or wounds. To differentiate between these two forms of resistance, natural infections can be reproduced by injecting inoculum through the silk channel, or by producing wounds using a needle, and introducing inoculum directly onto developing ears. Our protocol describes a technique used to inoculate developing maize ears with Fusarium graminearum, one of the fungal species that causes ear rot. We describe both silk channel and side needle inoculation techniques. Our protocol uses a backpack inoculator for both methods of infection, allowing for high-throughput inoculations, which are necessary for large field experiments. After harvest, the ears are visually rated on a percentage of disease scale. The protocol results in quantitative data that can be used for research on elucidating genetic resistance to fungal pathogens to assist breeding selections, and to understand plant-pathogen interactions of ear rots in maize.

Grain quality is defined as the suitability of grain for a particular use. It is usually designated by chemical composition or physical properties of the grain. The ability to measure grain quality is important for identity preservation of specialty grain market classes, for development of new varieties with improved quality through breeding, and for basic scientific studies on the genetic or biochemical control of grain quality traits. This review introduces official methods for measuring maize compositional traits, including protein, starch, oil, amino acid, phytate, and phosphorus content. Additionally, we discuss two nonofficial methods: measuring phytate and available phosphorus levels, and assessing amino acid balance. Phytate and available phosphorous impact the mineral nutrition of grain, while amino acid balance reflects the value of grain as a protein source and the bioavailability of protein. We also describe the use of near-infrared spectroscopy (NIRS) to assess levels of various compounds in maize. NIRS relies on the fact that compounds with differing molecular properties uniquely interact with the near-infrared region (750-2500 nm) of the electromagnetic radiation spectrum, and thus, generate spectral information that can be used to develop calibration models/equations for predicting the concentration of the compounds in grain samples. We discuss how sensitivity, accuracy, precision, throughput, and cost influence the choice of assay used to assess grain quality. Furthermore, we discuss how appropriate experimental design and data analysis can improve analytical outcomes when assessing grain quality.

Zea mays (maize) is a globally important cereal crop and a key system for studying plant development and stress responses. Proteome profiling and phosphoproteome profiling provide direct, quantitative readouts of protein abundance and phosphorylation states, which offer insights into aspects of regulation and cellular function that transcript-level measurements alone cannot provide. Robust and reproducible methods are essential for generating accurate and biologically relevant data in proteomics studies. The complexity of plant tissues, however, poses challenges for developing reliable sample preparation workflows. Here, we describe a detailed sample preparation protocol for quantitative proteome and phosphoproteome profiling in maize. The protocol encompasses protein extraction, filter-aided sample preparation (FASP), peptide desalting, tandem mass tag (TMT)-based labeling for quantitative multiplexing, and complementary TiO2 and Fe-NTA enrichment steps, yielding peptides suitable for analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). This approach enables the quantitative profiling of protein abundance and phosphorylation dynamics in maize tissues.

Plant hormones are small metabolites that regulate all aspects of plant growth and development, including plant defense. The detection and quantification of these hormones are critical to understanding the mechanism of growth regulation in plants. In maize, a wealth of genetic resources has enabled progress on elucidating the genetic mechanisms underlying plant growth. Biochemical studies of growth in maize can provide insight into the physiological mechanisms of growth control by measuring endogenous levels of plant hormones, and this knowledge would be enhanced by the development of a method to analyze several hormones in a single small sample of tissue. We provide here a simple protocol to extract and accurately quantify six classes of plant hormones in a single liquid chromatography/mass spectrometry injection run using maize tissues. Those hormones include abscisic acid (ABA), 1-aminocyclopropane-1-carboxylate (ACC), gibberellic acid (GA), 3-indoleacetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA), as well as an accumulated phytoanticipin of maize, 24-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which influences the levels of IAA.

Exogenous application of hormones in plants is a valuable technique for studying and manipulating plant growth, development, and responses to environmental stimuli. The foliar spray method is one of the most common approaches for the exogenous application of hormones in plants due to its ease of use on aerial organs (such as leaves and inflorescences) and the rapid absorption of the treated tissue, facilitating subsequent analyses. Here, we provide a protocol to implement this method in maize. The approach consists of preparing dilutions of the hormones or plant growth regulators (PGRs) of interest, usually in an aqueous solution and at low concentrations, followed by application by foliar spraying using a defined treatment regimen. Users can then evaluate effects by measuring different parameters, such as stem size, flowering time, seed production, or others. The foliar spray method can easily be scaled up and automated in greenhouse and field settings, and can be used to treat plants at all developmental stages.

Maize (Zea mays) is both an agronomically important crop and a reference model organism that has enabled the dissection of the molecular basis of plant development and environmental responses. Mass spectrometry-based proteomics provides a powerful approach to identify and quantify proteins and their post-translational modifications, facilitating the discovery of molecular mechanisms underlying complex biological processes. Unlike the study of gene expression using transcriptomics, analysis of the proteome and phosphoproteome provides direct measurement of proteins, which are responsible for driving or regulating nearly all cellular processes, thus offering a more complete picture of the cell's functional state. Over the past two decades, advancements in mass spectrometry have enabled large-scale profiling of protein abundance and phosphorylation sites in maize, improving our understanding of various biological phenomena. Here, we briefly summarize some of the major biological insights gained from maize proteome and phosphoproteome studies, and provide an overview of mass spectrometry sample preparation and acquisition/analysis workflows for the quantitative and reproducible analysis of protein abundance and phosphorylation dynamics in maize.

Maize is a globally important staple that is used as food for human and animal consumption, fuel, and other industrial applications. Pathogens affect all stages of the plant life cycle and every plant organ, and lead to significant yield losses. An integrated strategy incorporating cultural and chemical management practices, as well as development of resistant plant varieties, is needed to prevent yield losses due to plant diseases. Large numbers of breeding material must be screened to develop pathogen-resistant maize varieties. Inoculation methods must be high-throughput to accommodate the large screening experiments. Additionally, there needs to be an extensive understanding of the plant-pathogen interaction to use a targeted biotechnology-based approach, which takes advantage of knowledge of the system to engineer resistance. To evaluate germplasm for breeding and biotechnology approaches, inoculation methods must replicate natural infection, and disease severity must be rated consistently to accurately screen germplasm or gather data on pathogens of interest. Here, we review inoculation and rating methods for Gibberella ear rot, seedling blight caused by Globisporangium ultimum var. ultimum, and Goss's wilt that are efficient and high-throughput. We also introduce fluorescence microscopy techniques for leaf samples infected with Exserohilum turcicum, the causal agent of northern corn leaf blight. These pathogens all cause significant yield losses, and in particular, Gibberella ear rot is associated with the accumulation of harmful mycotoxins. Understanding how pathogens cause disease and how plants defend against attack is a major goal of maize pathology studies and critical for developing integrated management strategies.