Immunolocalization and Ultrastructure Show Ingestion of Cry Protein Expressed in by and Its Mode of Action.

Great interest exists in developing a transgenic trait that controls the economically important soybean () pest, soybean cyst nematode (SCN, ), due to its adaptation to native resistance. Soybean plants expressing the delta-endotoxin, Cry14Ab, were recently demonstrated to control SCN in both growth chamber and field testing. In that communication, ingestion of the Cry14Ab toxin by SCN second stage juveniles (J2s) was demonstrated using fluorescently labeled Cry14Ab in an in vitro assay.

Modes of action and potential as a peptide-based biofungicide of a plant defensin MtDef4.

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens.

A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium.

Volume electron microscopy technologies such as serial block face scanning electron microscopy (SBF-SEM) allow the characterization of tissue organization and cellular content in three dimensions at nanoscale resolution. Here, we describe the procedure to process and image an air-liquid interface culture of human or mouse airway epithelial cells for visualization of the multiciliated epithelium by SBF-SEM in vertical or horizontal cross section.

A conventional fixation volume electron microscopy protocol for plants.

Volume electron microscopy techniques play an important role in plant research from understanding organelles and unicellular forms to developmental studies, environmental effects and microbial interactions with large plant structures, to name a few. Due to large air voids central vacuole, cell wall and waxy cuticle, many plant tissues pose challenges when trying to achieve high quality morphology, metal staining and adequate conductivity for high-resolution volume EM studies. Here, we applied a robust conventional chemical fixation strategy to address the special challenges of plant samples and suitable for, but not limited to, serial block-face and focused ion beam scanning electron microscopy.

Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bio-inspired fungicide.

Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides.

Realizing the Full Potential of Advanced Microscopy Approaches for Interrogating Plant-Microbe Interactions.

Microscopy has served as a fundamental tool for insight and discovery in plant-microbe interactions for centuries. From classical light and electron microscopy to corresponding specialized methods for sample preparation and cellular contrasting agents, these approaches have become routine components in the toolkit of plant and microbiology scientists alike to visualize, probe and understand the nature of host-microbe relationships. Over the last three decades, three-dimensional perspectives led by the development of electron tomography, and especially, confocal techniques continue to provide remarkable clarity and spatial detail of tissue and cellular phenomena.

Metabolic synergy in Camelina reproductive tissues for seed development.

Photosynthesis in fruits is well documented, but its contribution to seed development and yield remains largely unquantified. In oilseeds, the pods are green and elevated with direct access to sunlight. With C labeling in planta and through an intact pod labeling system, a unique multi-tissue comprehensive flux model mechanistically described how pods assimilate up to one-half (33 to 45%) of seed carbon by proximal photosynthesis in .

Automated imaging of duckweed growth and development.

Duckweeds are the smallest angiosperms, possessing a simple body architecture and highest rates of biomass accumulation. They can grow near-exponentially via clonal propagation. Understanding their reproductive biology, growth, and development is essential to unlock their potential for phytoremediation, carbon capture, and nutrition.

A versatile enhanced freeze-substitution protocol for volume electron microscopy.

Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit.

Systems-wide analysis revealed shared and unique responses to moderate and acute high temperatures in the green alga Chlamydomonas reinhardtii.

Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35°C) and acute (40°C) high temperatures and subsequent recovery at 25°C. We identified previously overlooked unique elements in response to moderate high temperature.

Comparative Phenotyping of Two Commonly Used Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background).

The unicellular green alga is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves.

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content.

Organizing your space: The potential for integrating spatial transcriptomics and 3D imaging data in plants.

Plant cells communicate information for the regulation of development and responses to external stresses. A key form of this communication is transcriptional regulation, accomplished via complex gene networks operating both locally and systemically. To fully understand how genes are regulated across plant tissues and organs, high resolution, multi-dimensional spatial transcriptional data must be acquired and placed within a cellular and organismal context.

Time-Lapse Imaging of Root Pathogenesis and Fungal Proliferation Without Physically Disrupting Roots.

Microscopic observation of root disease onset and progression is typically performed by harvesting different plants at multiple time points. This approach prevents the monitoring of individual encounter sites over time, often mechanically damages roots, and exposes roots to unnatural conditions during observation. Here, we describe a method developed to avoid these problems and its application to study Fusarium oxysporum-Arabidopsis thaliana interactions.

X-ray microscopy enables multiscale high-resolution 3D imaging of plant cells, tissues, and organs.

Capturing complete internal anatomies of plant organs and tissues within their relevant morphological context remains a key challenge in plant science. While plant growth and development are inherently multiscale, conventional light, fluorescence, and electron microscopy platforms are typically limited to imaging of plant microstructure from small flat samples that lack a direct spatial context to, and represent only a small portion of, the relevant plant macrostructures. We demonstrate technical advances with a lab-based X-ray microscope (XRM) that bridge the imaging gap by providing multiscale high-resolution three-dimensional (3D) volumes of intact plant samples from the cell to the whole plant level.

High light and temperature reduce photosynthetic efficiency through different mechanisms in the C model Setaria viridis.

C plants frequently experience high light and high temperature conditions in the field, which reduce growth and yield. However, the mechanisms underlying these stress responses in C plants have been under-explored, especially the coordination between mesophyll (M) and bundle sheath (BS) cells. We investigated how the C model plant Setaria viridis responded to a four-hour high light or high temperature treatment at photosynthetic, transcriptomic, and ultrastructural levels.

Evaluation of multi-color genetically encoded Ca indicators in filamentous fungi.

Genetically encoded Ca indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca signaling in fungi, ranging from documenting Ca signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca indicators driven by multiple fungal promoters in Fusarium oxysporum, F.

Space: the final frontier - achieving single-cell, spatially resolved transcriptomics in plants.

Single-cell RNA-seq is a tool that generates a high resolution of transcriptional data that can be used to understand regulatory networks in biological systems. In plants, several methods have been established for transcriptional analysis in tissue sections, cell types, and/or single cells. These methods typically require cell sorting, transgenic plants, protoplasting, or other damaging or laborious processes.

Antifungal symbiotic peptide NCR044 exhibits unique structure and multifaceted mechanisms of action that confer plant protection.

In the indeterminate nodules of a model legume , ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens.

Roles of three Fusarium graminearum membrane Ca channels in the formation of Ca signatures, growth, development, pathogenicity and mycotoxin production.

Similar to animals and plants, external stimuli cause dynamic spatial and temporal changes of cytoplasmic Ca in fungi. Such changes are referred as the Ca signature and control cellular responses by modulating the activity or location of diverse Ca-binding proteins (CBPs) and also indirectly affecting proteins that interact with CBPs. To understand the mechanism underpinning Ca signaling, therefore, characterization of how Ca moves to and from the cytoplasm to create Ca signatures under different conditions is fundamental.

Optimization of the HyPer sensor for robust real-time detection of hydrogen peroxide in the rice blast fungus.

Reactive oxygen species (ROS) production and breakdown have been studied in detail in plant-pathogenic fungi, including the rice blast fungus, Magnaporthe oryzae; however, the examination of the dynamic process of ROS production in real time has proven to be challenging. We resynthesized an existing ROS sensor, called HyPer, to exhibit optimized codon bias for fungi, specifically Neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct could detect changes in fungal ROS during the plant infection process. Using confocal microscopy, we were able to visualize fluctuating ROS levels during the formation of an appressorium on an artificial hydrophobic surface, as well as during infection on host leaves.

Super-Resolution Microscopy: From Single Molecules to Supramolecular Assemblies.

Super-resolution microscopy (SRM) methods have allowed scientists to exceed the diffraction limit of light, enabling the discovery and investigation of cellular structures at the nanometer scale, from individual proteins to entire organelles. In this review we survey the application of SRM in elucidating the structure of macromolecules in the native cellular environment. We emphasize how SRM can generate molecular maps of protein complexes and extract quantitative information on the number, size, distribution, and spatial organization of macromolecules.

Roles of three Fusarium oxysporum calcium ion (Ca(2+)) channels in generating Ca(2+) signatures and controlling growth.

Spatial and temporal changes of cytoplasmic calcium ions ([Ca(2+)]c), caused by external stimuli, are known as the Ca(2+) signature and presumably control cellular and developmental responses. Multiple types of ion channels, pumps, and transporters on plasma and organellar membranes modulate influx and efflux of Ca(2+) to and from the extracellular environment and internal Ca(2+) stores to form Ca(2+) signatures. Expression of a fluorescent protein-based Ca(2+) probe, Cameleon YC3.