Single-Molecule Imaging of Transport through Plasmodesmata in Live Plants

Objective

Plants coordinate growth and environmental responses through nanoscale channels called plasmodesmata (PD), which allow proteins, RNA, hormones, and metabolites to pass directly between cells. Current PD transport assays average fluxes across the hundreds of channels that connect a cell to its neighbors, obscuring their heterogeneity and regulation. As a result, the real-time activity of individual PD has never been directly observed. PLANTOSCOPE will establish Transport Localization Microscopy (TLM), a super-resolution imaging strategy that converts transient single-molecule passages through PD into quantitative 3D transport maps with ~10 nanometer and ~10 millisecond precision in living plants. TLM combines 3D single-molecule localization microscopy (SMLM) with probes engineered to yield resolvable passage times. 3D SMLM will be achieved through point spread function (PSF) engineering, and probes will include genetically encoded fluorescent proteins, labeled DNA/RNA strands and small dextrans, and ultrashort fluorescent carbon nanotubes. Analysis will extract per-PD passage times and coordinates in Arabidopsis thaliana roots, enabling real-time readouts from thousands of channels in situ. These passage-time distributions will provide unprecedented insights into confined transport mechanisms within PD. Using TLM, we will quantify how genetic and hormonal regulators reshape single-PD activity and passage statistics, compare connectivity across cell types and tissues, and track shifts under biotic and abiotic stress to reveal early signatures before visible symptoms. The project is hosted jointly at CNRS/Univ. Bordeaux by teams specializing in single-molecule microscopy and PD biology. By delivering the first functional single-molecule assay of intercellular transport in living plants, PLANTOSCOPE will resolve PD heterogeneity and regulation in vivo and clarify how PD dynamics modulate intercellular connectivity under pathogen and climate-related stress.