Biology subjects

Burch-Smith, T. M.

Publications and source records attributed to Burch-Smith, T. M..

7 recordsLinked to original sources

bioRxiv2026

The Application of 3D Cryo-Volume Electron Microscopy to Visualize and Quantify the Photosynthetic Filamentous Cyanobacterium Anabaena

Cryogenic volume electron microscopy (cryo-vEM) is a rapidly developing nanoscale imaging approach that enables three-dimensional datasets of vitrified cells and tissues at intermediate resolutions without treatment with chemical fixatives or metal contrasting agents. Our goal was to evaluate the suitability of cryo-vEM for whole cells of photosynthetic organisms such as cyanobacteria. Two species of Anabaena, a filamentous cyanobacterium, were captured on poly-L-lysine-coated grids, vitrified by plunge-freezing into liquid ethane, and mapped by secondary electron and integrated fluorescence light microscopy to identify target cells. Cells of interest were coated with platinum, trenched with xenon, and 3D volumes collected by alternating argon milling and through-lens detector (TLD) imaging on a Thermo Scientific Hydra plasma focused ion beam scanning electron microscope. Volumes were segmented via 2.5D U-Net models, then quantified in Dragonfly 3D World. Block-face images revealed the cell wall, thylakoids, phycobilisomes, carboxysomes, cyanophycin and polyphosphate granules. Whole-cell 3D rendering allowed quantification of carboxysomes, cyanophycin granules, and measurement of thylakoid thickness and spacing in A. inaequalis and Anabaena sp. UTEX 2576. Thus, cryo-vEM provided interpretable whole-cell ultrastructure of cyanobacteria without chemical pre-treatment, and its compatibility with cryo-fluorescence and in situ cryo-ET enables contextual multi-scale perspectives from whole cells to molecular structures.

cell biology↗

bioRxiv2026

High-resolution microscopy of whole plant cells reveals virus-induced changes to intercellular connectivity

Plasmodesmata (PD) provide plant viruses a direct route for cell-to-cell spread. Viral infection increases intercellular trafficking via PD, termed gating, but the mechanism involved remains unclear. Here, using a combination of high-resolution volume electron microscopy and live cell imaging, we demonstrate that diverse viruses increased the number of PD in infected leaves. This increase was triggered by viral movement proteins and the induction of PD formation depended on the presence of the virus and the localization of movement proteins to PD. Further, Group I Remorins, known inhibitors of virus infection, are negative regulators of PD formation and inhibit virus or movement protein-induced changes to PD density. Our results lead to a model in which viral gating of PD may result from increased de novo PD formation.

plant biology↗

bioRxiv2026

Reprogramming of auxin and brassinosteroid signaling is an early part of the homeostatic response to a viral movement protein

Plant viruses rely on intercellular trafficking via plasmodesmata (PD) to move between cells in their hosts. This ability is conferred by virus encoded movement proteins (MPs), which can increase plasmodesmal permeability and intercellular trafficking independent of other viral proteins. Callose dynamics in the cell walls surrounding PD have a critical role in determining plasmodesmal flux, with decreased callose levels correlates with increased trafficking. Notably, PD callose levels are both increased and decreased during virus infection, suggesting that there are regulatory responses to the virus. Here, we found that auxin and brassinosteroid (BR) exert opposing effects on PD connectivity. While auxin enhances intercellular trafficking primarily by promoting PD density, BR restricts connectivity by reducing reduced PD biogenesis and increasing callose accumulation. We identified genes involved in auxin and BR signaling as Most of those genes encode membrane-associated proteins. We identified the receptor-like protein RLP15 as a critical upstream regulator of intracellular auxin homeostasis through stabilization of the ER-localized auxin transporter PILS5. In parallel, negative regulators of PD permeability including ERECTA, PPI, CER3, and DEAL2 define a host connectivity restraint network. These findings point to an auxin-BR module as a nexus for determining the degree of changes in plasmodesmal permeability that is elicited by the viral MP. Together with changes in callose dynamics at PD, this regulatory node allows plants to maintain homeostasis of intercellular trafficking, possibly contributing to maintenance of cell and tissue integrity during infection. Significance StatementPlant viruses encode movement proteins (MP) which increase plasmodesmal permeability to allow the local cell-to-cell trafficking of viral entities. This study identifies a non-canonical auxin-BR antagonistic module that regulates plasmodesmal connectivity in response to the changes triggered by viral movement protein early in the infection cycle. We demonstrate that MP30, encoded by the tobacco mosaic virus, rewires BRs and auxins roles in controlling intercellular communication by interfering with membrane proteins associated with these hormonal pathways. By defining this hormonal nexus, our findings reveal a sophisticated host-pathogen interface where plants attempt to maintain intercellular homeostasis during the onset of viral pathogenesis.

plant biology↗

bioRxiv2025

Heme acts a chloroplast-to-nucleus retrograde signal to regulate intercellular trafficking via plasmodesmata

Intercellular communication via plasmodesmata (PD) is essential for plant growth, development, and defense, yet its regulation remains poorly understood. Chloroplasts communicate information about the environment and the physiological state of the plant cell to the nucleus. It has been proposed that chloroplast-generated signals also modulate the expression of nuclear genes related to PD to regulate the trafficking of photosynthetic products and metabolites along with other molecules that act non-cell-autonomously. In this study, we set out to identify the chloroplast retrograde signals that regulate intercellular trafficking via PD. Using a combination of Arabidopsis thaliana mutants and gene silencing in Nicotiana benthamiana, we found that the metabolites of the tetrapyrrole biosynthetic pathway, most likely heme, can act to modulate PD-mediated intercellular trafficking. We also identified genes that are potentially regulated by the heme signal to modify plasmodesmata function. Together, these findings strengthen the link between chloroplasts and PD in coordinating intercellular communication for optimal plant development and resource allocation.

plant biology↗

bioRxiv2024

Structurally diverse calloses/β-1,3-glucans in plant cell wall microdomains

Cell walls underpin the mechanics of cell growth, intercellular signalling, and defence against pathogenic organisms. {beta}-(1,3)-glucans (also known as callose) are polysaccharides found in plants, fungi, and some bacterial species. In developing plant organs, callose accumulates around intercellular channels (plasmodesmata) controlling cell-to-cell communication. We developed monoclonal antibodies for the detection of {beta}-(1,3)-glucans and using these identified distinct populations of callose differing in size and secondary structure. Callose sub-populations were in proximal but not overlapping cell wall microdomains implying distinct spatial and functional microenvironments. We also unveiled callose interaction with xyloglucan; another plant glycan regulating cell wall functions. This work challenges previous views demonstrating structural heterogeneity in plant callose and supporting interactions between glycans with roles in the regulation of cell wall properties and functions.

plant biology↗

bioRxiv2023

Reliable detection and quantification of plasmodesmal callose in Nicotiana benthamiana leaves during defense responses

Callose, a beta-(1,3)-D-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD, or conversely by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during innate immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing the intercellular trafficking activity during plant immunity. Despite the popularity of this metric there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying PD callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescent microscopy to measure callose deposition in fixed tissue. Manual or semi-automated workflows for image analysis were also compared and found to produce similar results although the semi-automated workflow produced a wider distribution of data points.

plant biology↗

bioRxiv2021

Mathematical modeling suggests cooperation of plant-infecting viruses

Viruses are major pathogens of agricultural crops. Viral infections often start after the virus enters the outer layer of a tissue or surface and many successful viruses, after local replication in the infected tissue, are able to spread systemically. Quantitative details of virus dynamics in plants, however, have been poorly understood, in part, because of the lack of experimental methods allowing to accurately measure the degree of infection in individual plant tissues. Recently, by using flow cytometry and two different flourescently-labeled strains of the Tobacco etch virus (TEV), Venus and BFP, kinetics of viral infection of individual cells in leaves of Nicotiana tabacum plants was followed over time [1]. A simple mathematical model, assuming that viral spread occurs from lower to upper leaves, was fitted to these data. While the the original model could accurately describe the kinetics of viral spread locally and systemically, we also found that many alternative versions of the model, for example, if viral spread starts at upper leaves and progresses to lower leaves or when virus dissemination is stopped due to an immune response, provided fits of the data with reasonable quality, and yet with different parameter estimates. These results strongly suggest that experimental measurements of the virus infection in individual leaves may not be sufficient to identify the pathways of viral dissemination between different leaves and reasons for viral control; we propose experiments that may allow discrimination between the alternatives. By analyzing the kinetics of coinfection of individual cells by Venus and BFP strains of TEV we found a strong deviation from the random infection model, suggesting cooperation between the two strains when infecting plant cells. Importantly, we showed that many mathematical models on the kinetics of coinfection of cells with two strains could not adequately describe the data, and the best fit model needed to assume i) different susceptibility of uninfected cells to infection by two viruses locally in the leaf vs. systemically from other leaves, and ii) decrease in the infection rate depending on the fraction of uninfected cells which could be due to a systemic immune response. Our results thus demonstrate the difficulty in reaching definite conclusions from extensive and yet limited experimental data and provide evidence of potential cooperation between different viral variants infecting individual cells in plants.

microbiology↗