Avian eggshell membrane (ESM) is fabricated within the isthmus region of the oviduct and is comprised of three juxtaposed, predominantly proteinaceous layers lying between egg white and the calcified shell. The limiting membrane is less than 0.5 μm in thickness and forms the osmotic barrier for the egg. This first layer provides the foundation for the successive deposition of two mats of protein fibers. Fibers from both inner and outer layers appear to have similar amino acid compositions and are notably disulfide-rich (comprising about 10% Cys). ESM has been utilized in a wide variety of applications, including nutraceutical supplements, tissue engineering, and nanofabrication, and yet fundamental questions concerning protein composition, fiber structure, and membrane assembly remain to be resolved. We previously identified an abundant disulfide-rich structural protein in chicken ESM fibers (cysteine-rich eggshell membrane protein; CREMP) that contains multiple tandemly repeated modules. In this work, we determine a structural model for four consecutive 2-disulfide containing CREMP modules using a variety of two- and three-dimensional solution NMR experiments. CREMP modules feature an N-terminal loop region positioned above a small beta hairpin that is stabilized by a conserved pattern of disulfide bridges between Cys1-3 and Cys2-4. While the individual CREMP modules are highly ordered, the lack of long-range inter-module restraints suggests an extended structure connected by flexible linkers. Finally, the structural information obtained in this work is considered in the context of full-length CREMP proteins and compared to two other structural proteins that contain multiple tandem repeats of 2-disulfide modules.

Quantitative disease resistance in plants emerges from complex interactions between host tissues and pathogen growth dynamics, producing a spectrum of phenotypic responses. In plant-fungal interactions, disease is most visibly expressed through lesions that vary in number, size, shape, and color, collectively defining a lesion profile. For Cochliobolus heterostrophus, a fungus causing Southern Corn Leaf Blight of maize (Zea mays ssp. mays), we show that infection on different maize genotypes produces strikingly different lesion profiles. However, it remains unclear whether such macroscopic variation in lesion profiles corresponds to consistent differences in the three-dimensional organization of pathogen colonization within host tissue. We therefore examined variation in the three-dimensional structure of C. heterostrophus-infection networks across host genotypes representing four lesion-profile classes. Using light-sheet microscopy and filament-tracing methods adapted from neuroscience, we developed quantitative metrics to characterize infection network organization, including depth, density, shape, and spatial association with host vascular tissue. In this dataset, network depth was similar across genotypes, whereas network morphology (shape and density), spatial association with vascular bundles, hyphal segment length, and branching frequency varied. Notably, genotypes with similar quantitative resistance levels sometimes exhibited distinct patterns of fungal colonization, suggesting that comparable resistance can arise from different underlying infection dynamics. These findings indicate that lesion profiles may not uniquely predict infection network structure and highlight the utility of three-dimensional network metrics for describing variation that likely reflects multiple underlying host and pathogen processes. This multi-scale framework provides tools for linking macroscopic disease phenotypes with microscopic infection processes in quantitative disease resistance.

Hydrogen peroxide (H2O2) is a potent reactive oxygen species that plays a crucial role as a versatile signaling molecule for cellular function and vitality. Recent experimental evidence indicates that H2O2 affects cell-to-cell communication through plasmodesmata, tiny cytoplasmic nanopores connecting adjacent plant cells. H2O2-dependent systemic signaling has also been reported to involve plasmodesmal function in some contexts, although the dominant routes and messengers underlying rapid long-distance signaling remain under active debate. Nevertheless, direct monitoring of redox dynamics at plasmodesmata in live tissues has remained challenging. In this study, we developed a plasmodesmata-localized HyPer7 (Pd-HyPer7) reporter to investigate H2O2 dynamics at plasmodesmata in response to exogenous redox stressors and plant stresses, including cold and mechanical wounding. Pd-HyPer7 showed response characteristics that differed from the HyPer7 reporters localized to the cytosol, plasma membrane, and chloroplasts under the conditions tested, indicating that redox responses at plasmodesmata are distinguishable from these compartments. Notably, during mechanical wounding, both the cytosol and plasmodesmata showed transient redox responses with broadly similar temporal profiles in local tissues. In systemic tissues, however, the responses were temporally separated, with plasmodesmal oxidation peaking well after the cytosolic response. This timing relationship is consistent with plasmodesmata acting downstream of early systemic wound signaling, rather than simply mirroring cytosolic redox dynamics. Together, our results establish Pd-HyPer7 as a tool for monitoring plasmodesmal redox dynamics and support a model in which plasmodesmata participate in spatially and temporally regulated redox responses during plant stress.

While liquid chromatography-mass spectrometry (LCMS) has revolutionized plant proteomics over the past decade, plant sample preparation remains a major challenge due to rigid cell walls, abundant secondary metabolites, and wide dynamic range of protein abundance. These hurdles demand laborious tissue disruption, complex precipitation, and extensive cleanup prior to LCMS analysis, limiting the widespread adoption of proteomic technologies within the plant biology community. To overcome these barriers, we introduced an "in-cell proteomics" strategy that bypasses cell lysis and protein extraction by performing digestion directly inside methanol-fixed cells. We systematically benchmarked this strategy against conventional lysate-based workflows across 4 model plants (Arabidopsis thaliana, Nicotiana benthamiana, Zea mays, and Sorghum bicolor) and 3 tissue types (leaves, pollen, and seeds). Combined with minimal input material and single-shot LCMS, the in-cell approach consistently identified 9,000 to 12,000 proteins from leaves, 7,000 to 9,000 from pollen grains, and approximately 8,000 from seeds. Our comprehensive dataset demonstrates that this in-cell digestion approach substantially simplifies plant sample preparation while delivering proteomic performance equivalent to established workflows. Finally, to demonstrate the biological utility of this approach, we characterized the proteomes of N. benthamiana leaves infected with 2 fungal strains that exhibit different host specificities. Our in-depth proteomic data revealed distinct host response signatures differentiating the host-adapted Colletotrichum destructivum from the nonhost-adapted Colletotrichum sublineola strain. Overall, this study provides a simple, unbiased alternative for plant proteomic analysis that can be readily applied to tackle complex agricultural and physiological challenges in plant biology.

Although modern mass spectrometry (MS) based proteomics has been broadly employed by the plant science community, proteomics analysis of plant tissues remains highly challenging to general plant biology laboratories, mainly due to the lack of effective and easily adaptable strategies that can process plant samples for MS analysis rapidly and reliably. Existing methods often involves lengthy mechanical disruption and protein extraction, which not only require large starting materials but also can cause significant variations, compromising quantitation accuracy. Here, we present an "in-cell proteomics" strategy and benchmark its performance against conventional processing methods using several model plants, including Arabidopsis thaliana, Nicotiana benthamiana, Zea mays and Sorghum bicolor. The comprehensive datasets from this study indicate that the on-filter in-cell (OFIC) processing-based "in-cell proteomics" strategy can eliminate cell lysis of plant leaves and pollen grains, and process proteins directly, enabling the identification of 9,000 to 12,000 proteins from leaves, and 7,000 to 9,000 proteins from pollen grains using one-shot LCMS. The OFIC strategy demonstrated broadly equivalent or better performance over conventional approaches in the context of simplicity, speed, digestion effectiveness, and quantitation accuracy. Applying the strategy to investigate plant leaf infection of N. benthamiana leaves revealed interesting signature proteins that respond differentially to host-adapted Colletotrichum destructivum and nonhost-adapted Colletotrichum sublineola fungus. The single-vessel approach bypasses cell disruption, substantially simplifying plant sample preparation while reducing the required sample input and enhancing proteomic sensitivity. It offered a generalizable approach to study plant leaf infection and other significant questions in plant biology and beyond.

A U-Net machine learning algorithm was adapted to automatically segment tendon collagen fibril cross-sections from serial block face scanning electron microscopy (SBF-SEM) and create three-dimensional (3D) renderings. We compared the performance of routine Otsu thresholding and U-Net for a positional tendon that has low fibril density (rat tail tendon), an energy-storing tendon that has high fibril density (rat plantaris tendon), and a high fibril density tendon hypothesized to have disorganized 3D ultrastructure (degenerated rat plantaris tendon). The area segmentation of the tail and healthy plantaris tendon had excellent accuracy for both the Otsu and U-Net, with an Intersection over Union (IoU) of 0.8. With degeneration, only the U-Net could accurately segment the area, whereas Otsu IoU was only 0.45. For boundary validation, the U-Net outperformed Otsu segmentation for all tendons. The fibril diameter from U-Net was within 10% of the manual segmentation, however, the Otsu underestimated the fibril diameter by 39% in healthy plantaris and by 84% in the degenerated plantaris. Fibril geometry was averaged across the entire image stack and compared across tendon types. The tail had a lower fibril area fraction (58%) and larger fibril diameter (0.31 µm) than the healthy plantaris (67% and 0.21 µm) and degenerated plantaris tendon (66% and 0.19 µm). This method can be applied to a large variety of tissues to quantify 3D collagen fibril structure.

Extracellular vesicles are membrane-bound organelles that play crucial roles in intercellular communication and elicit responses in the recipient cell, such as defense responses against pathogens. In this study, we have optimized a protocol for isolating extracellular vesicles (EVs) from Sorghum bicolor apoplastic wash. We characterized the EVs using fluorescence microscopy and correlative light and electron microscopy.

Plant small RNAs are 21-24 nucleotide, noncoding RNAs that function as regulators in plant growth and development. Colorimetric detection of plant small RNAs was made possible with the introduction of locked nucleic acid probes. However, fluorescent detection of plant small RNAs has been challenging due to the high autofluorescence from plant tissue. Here we report a fluorescent in situ detection method for plant small RNAs. This method can be applied to most plant samples and tissue types and also can be adapted for single-molecule detection of small RNAs with super-resolution microscopy.

Chloroplast Unusual Positioning 1 (CHUP1) plays an important role in the chloroplast avoidance and accumulation responses in mesophyll cells. In epidermal cells, prior research showed silencing CHUP1-induced chloroplast stromules and amplified effector-triggered immunity (ETI); however, the underlying mechanisms remain largely unknown. CHUP1 has a dual function in anchoring chloroplasts and recruiting chloroplast-associated actin (cp-actin) filaments for blue light-induced movement. To determine which function is critical for ETI, we developed an approach to quantify chloroplast anchoring and movement in epidermal cells. Our data show that silencing NbCHUP1 in Nicotiana benthamiana plants increased epidermal chloroplast de-anchoring and basal movement but did not fully disrupt blue light-induced chloroplast movement. Silencing NbCHUP1 auto-activated epidermal chloroplast defense (ECD) responses including stromule formation, perinuclear chloroplast clustering, the epidermal chloroplast response (ECR), and the chloroplast reactive oxygen species (ROS), hydrogen peroxide (H2O2). These findings show chloroplast anchoring restricts a multifaceted ECD response. Our results also show that the accumulated chloroplastic H2O2 in NbCHUP1-silenced plants was not required for the increased basal epidermal chloroplast movement but was essential for increased stromules and enhanced ETI. This finding indicates that chloroplast de-anchoring and H2O2 play separate but essential roles during ETI.

Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.

In plants, cytoplasmic mRNA decay is critical for posttranscriptionally controlling gene expression and for maintaining cellular RNA homeostasis. Arabidopsis DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1) is a cytoplasmic mRNA decay factor that interacts with proteins involved in mRNA decapping and nonsense-mediated mRNA decay (NMD). There is limited information on the functional role of DNE1 in RNA turnover, and the identities of its endogenous targets are unknown. In this study, we utilized RNA degradome approaches to globally investigate DNE1 substrates. Monophosphorylated 5 ' ends, produced by DNE1, should accumulate in mutants lacking the cytoplasmic exoribonuclease XRN4, but be absent from DNE1 and XRN4 double mutants. In seedlings, we identified over 200 such transcripts, most of which reflect cleavage within coding regions. While most DNE1 targets were NMD-insensitive, some were upstream ORF (uORF)-containing and NMD-sensitive transcripts, indicating that this endoribonuclease is required for turnover of a diverse set of mRNAs. Transgenic plants expressing DNE1 cDNA with an active-site mutation in the endoribonuclease domain abolished the in planta cleavage of transcripts, demonstrating that DNE1 endoribonuclease activity is required for cleavage. Our work provides key insights into the identity of DNE1 substrates and enhances our understanding of DNE1-mediated mRNA decay.

Plant extracellular vesicles (EVs) are membrane bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. In this paper, we described the isolation and characterization of extracellular vesicles from Sorghum bicolor. Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, Calcein-acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also being found in the EV proteome of Arabidopsis. These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium. Here, we discover the conserved ion channel CLHM-1 as a ciliary EV cargo. Imaging of EVs released from sensory neuron cilia of Caenorhabditis elegans expressing fluorescently tagged CLHM-1 and TRP polycystin-2 channel PKD-2 shows enrichment of these cargoes in distinct EV subpopulations that are differentially shed in response to mating partner availability. PKD-2 alone is present in EVs shed from the cilium distal tip, whereas CLHM-1 EVs bud from a secondary site(s), including the ciliary base. Heterotrimeric and homodimeric kinesin-2 motors have discrete impacts on PKD-2 and CLHM-1 colocalization in both cilia and EVs. Total loss of kinesin-2 activity decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde intraflagellar transport is required for selective enrichment of protein cargoes into heterogeneous EVs with different signaling potentials.

Summary The most abundant cellular divalent cations, Mg2+ (mM) and Ca2+ (nM-μM), antagonistically regulate divergent metabolic pathways with several orders of magnitude affinity preference, but the physiological significance of this competition remains elusive. In mice consuming a Western diet, genetic ablation of the mitochondrial Mg2+ channel Mrs2 prevents weight gain, enhances mitochondrial activity, decreases fat accumulation in the liver, and causes prominent browning of white adipose. Mrs2 deficiency restrains citrate efflux from the mitochondria, making it unavailable to support de novo lipogenesis. As citrate is an endogenous Mg2+ chelator, this may represent an adaptive response to a perceived deficit of the cation. Transcriptional profiling of liver and white adipose reveals higher expression of genes involved in glycolysis, β-oxidation, thermogenesis, and HIF-1α-targets, in Mrs2−/− mice that are further enhanced under Western-diet-associated metabolic stress. Thus, lowering mMg2+ promotes metabolism and dampens diet-induced obesity and metabolic syndrome.

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.

Filamentous objects are ubiquitous in biomedical images, and segmenting individual filaments is fundamental for biomedical research. Unlike common objects with well-defined boundaries and centers, filaments are thin, non-rigid, varying in shape, and often densely overlapping. These properties make it extremely challenging to extract individual filaments. This paper proposes a novel approach to extract filamentous objects by transforming an instance segmentation problem into a sequence modeling problem. Our approach first identifies filaments' tip points, and we segment each instance by tracing them from each tip with a sequential encoder-decoder framework. The proposed method simulates the process of humans extracting filaments: pick a tip and trace the filament. As few datasets contain instance labels of filaments, we first generate synthetic filament datasets for training and evaluation. Then, we collected a dataset of 15 microscopic images of microtubules with instance labels for evaluation. Our proposed method can alleviate the data shortage problem since our proposed model can be trained with synthetic data and achieve state-of-art results when directly evaluated on the microtubule dataset and P. rubescens dataset. We also demonstrate our approaches' capabilities in extracting short and thick elongated objects by evaluating on the C. elegans dataset. Our method achieves a comparable result compared to the state-of-art method with faster processing time. Our code is available at https://github.com/VimsLab/DRIFT .