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1. Tetrapyrrole biosynthetic intermediates act as chloroplast‐to‐nucleus retrograde signals to regulate intercellular trafficking via plasmodesmata

   https://doi.org/10.1111/nph.70978  

   Azim, Mohammad F; Gifford, Levi B; Alazem, Mazen; Woodson, Jesse D; Burch‐Smith, Tessa M (April 2026, New Phytologist)

   Summary Intercellular communication via plasmodesmata (PD) is essential for plant growth, development, and defense, yet its regulation remains poorly understood. We set out to identify the chloroplast retrograde signals that regulate intercellular trafficking via PD.Using a combination ofArabidopsis thalianamutants and gene silencing inNicotiana benthamiana, we found that the metabolites of the tetrapyrrole biosynthetic pathway, most likely heme, can act to modulate PD‐mediated intercellular trafficking. Knockout or knockdown ofGUN5, encoding the catalytic subunit of the magnesium chelatase, revealed a correlation between reduced levels of total heme and severely decreased intercellular trafficking.Further, plants with reduced expression of genes involved in chloroplast RNA processing were shown to have decreased total heme levels and decreased intercellular trafficking. Like withGUN5, reduced expression ofFC2, but notFC1, led to reduced levels of total heme and drastically reduced intercellular trafficking.Thus, the results support a model where a specific pool of heme regulates plasmodesmata and intercellular trafficking. We also identified genes that are potentially regulated by the heme signal to modify plasmodesmal function. Together, these findings strengthen the link between chloroplasts and PD in coordinating intercellular communication for optimal plant development and resource allocation. 

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2. Glucosinolates can act as signals to modulate intercellular trafficking via plasmodesmata

   https://doi.org/10.1111/nph.70032  

   Fernandez, Jessica C; Azim, Mohammad F; Adams, Nicole; Strong, Morgan; Piya, Sarbottam; Xu, Min; Brunkard, Jacob O; Hewezi, Tarek; Sams, Carl E; Burch‐Smith, Tessa M (May 2025, New Phytologist)

   Summary Plasmodesmata (PD) allow direct communication across the cellulosic plant cell wall, facilitating the intercellular movement of metabolites and signaling molecules within the symplast. InArabidopsis thalianaembryos with reduced levels of the chloroplast RNA helicase ISE2, intercellular trafficking and the number of branched PD were increased. We therefore investigated the relationship between alteredISE2expression and intercellular trafficking.Gene expression analyses in Arabidopsis tissues whereISE2expression was increased or decreased identified genes associated with the metabolism of glucosinolates (GLSs) as highly affected.Concomitant with changes in the expression of GLS‐related genes, plants with abnormalISE2expression contained altered GLS metabolic profiles compared with wild‐type (WT) counterparts. Indeed, changes in the expression of GLS‐associated genes led to altered intercellular trafficking in Arabidopsis leaves. Exogenous application of GLSs but not their breakdown products also resulted in altered intercellular trafficking.These changes in trafficking may be mediated by callose levels at PD as exogenous GLS treatment was sufficient to modulate plasmodesmal callose in WT plants. Furthermore, auxin metabolism was perturbed in plants with increased indole‐type GLS levels. These findings suggest that GLSs, which are themselves transported between cells via PD, can act on PD to regulate plasmodesmal trafficking capacity. 

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3. Measuring Plasmodesmata‐mediated Intercellular Trafficking Using Microparticle Bombardment in Arabidopsis and Crops

   https://doi.org/10.1002/cpz1.70194  

   Li, Zhongpeng; Thorpe, Connor; Jiang, Shan; Aung, Kyaw (August 2025, Current Protocols)

   Abstract Plasmodesmata (PD) are highly specialized, nanoscopic pores that traverse the cell wall to connect the cytoplasm of adjacent plant cells, enabling direct cell‐to‐cell communication. PD provides the continuity of three key cellular components: the plasma membrane, the endoplasmic reticulum (ER), and the cytosol. The compressed ER within PD is known as the desmotubule. PD mediates the intercellular trafficking of ions, metabolites, hormones, proteins, and RNA molecules between adjacent cells. Although several methods have been developed to quantify PD‐mediated molecular trafficking, it remains a technical challenge. Among these, PD‐mediated movement of fluorescent proteins is one of the most commonly used approaches. Here we present a microparticle bombardment method using a biolistic particle delivery system to investigate the PD‐mediated movement of fluorescent proteins. We equipped the delivery system with a flow guiding barrel to improve bombardment efficiency and consistency. We demonstrated the effects of gold particle aggregation and plant age on transformation efficiency and protein movement inArabidopsis. We also showed the feasibility of the method in determining PD‐mediated movement in tomato, pepper, and soybean. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Microparticle bombardment assay for measuring plasmodesmata‐mediated trafficking 

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

   https://doi.org/10.64898/2026.02.14.705926  

   Alazem, Mazen; Kreder, Jessi; Baldrich, Patricia; Nuzzi, Samantha P; Burch-Smith, Tessa M (February 2026, bioRxiv)

   Abstract 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 BR’s and auxin’s 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. 

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5. Comparing methods for detection and quantification of plasmodesmal callose in Nicotiana benthamiana leaves during defense responses

   https://doi.org/10.1094/MPMI-09-23-0152-SC  

   Sankoh, Amie Fornah; Adjei, Joseph; Roberts, Daniel; Burch-Smith, Tessa M. (February 2024, Molecular Plant-Microbe Interactions®)

   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 immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing likely 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, we compared 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. 

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