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Abstract Plasmodesmata (PD) are essential cellular structures that facilitate intercellular communication in plants, enabling the transport of nutrients and signaling molecules. Over the past decades, significant strides have been made in unraveling the formation, function, and regulation of PD. Identification and functional characterization of PD-associated proteins have greatly advanced our understanding of PD. This review discusses past efforts in uncovering PD proteomes and highlights recent breakthroughs in applying proximity labeling (PL) assays to map plant protein interactomes. Special attention is given to using PL assays in studying PD biology, emphasizing their potential to drive future advancements and deepen our understanding of PD function and regulation. By integrating PL technologies with established methodologies, researchers can gain comprehensive insights into the dynamic composition and roles of PD. 

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 

Abstract Plasmodesmata connect adjoining plant cells, allowing molecules to move between the connected cells for communication and sharing resources. It has been well established that the plant polysaccharide callose is deposited at plasmodesmata, regulating their aperture and function. Among proteins involved in maintaining callose homeostasis, PLASMODESMATA-LOCATED PROTEINSs (PDLPs) promote callose deposition at plasmodesmata. This study explored the function of PDLP5 and PDLP6 in different cell types. We discovered that PDLP5 and PDLP6 are expressed in nonoverlapping cell types in Arabidopsis (Arabidopsis thaliana). The overexpression of PDLP5 and PDLP6 results in the overaccumulation of plasmodesmal callose at different cell interfaces, indicating that PDLP5 and PDLP6 are active in different cell types. We also observed 2 distinct patterns of starch accumulation in mature leaves of PDLP5 and PDLP6 overexpressors. An enzyme-catalyzed proximity labeling approach was used to identify putative functional partners of the PDLPs. We identified SUCROSE SYNTHASE 6 (SUS6) as a functional partner of PDLP6 in the vasculature. We further demonstrated that PDLP6 physically and genetically interacts with SUS6. In addition, CALLOSE SYNTHASE 7 (CALS7) physically interacts with SUS6 and PDLP6. Genetic interaction studies showed that CALS7 is required for PDLP6 function. We propose that PDLP6 functions with SUS6 and CALS7 in the vasculature to regulate plasmodesmal function.