Assaying effector cell-to-cell mobility in plant tissues identifies hypermobility and indirect manipulation of plasmodesmata

In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. While plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To establish a methodology for identification and characterization of the cell-to-cell mobility of effectors, we performed a quantitative live imaging-based screen of candidate effectors of the fungal pathogen Colletotrichum higginsianum. We predicted C. higginsianum effectors by their expression profiles, the presence of a secretion signal, and their predicted and in planta localization when fused to GFP. We assayed for cell-to-cell mobility of nucleo-cytosolic effectors and identified 14 that are cell-to-cell mobile. We identified that 3 of these effectors are “hypermobile”, showing cell-to-cell mobility greater than expected for a protein of its size. To explore the mechanism of hypermobility we chose two hypermobile effectors and measured their impact on plasmodesmata function and found that even though they show no direct association with plasmodesmata, each increases the transport capacity of plasmodesmata. Thus, our methods for quantitative analysis of cell-to-cell mobility of candidate microbe-derived36effectors, or any suite of host proteins, can identify cell-to-cell hypermobility and offer greater understanding of how proteins affect plasmodesmal function and intercellular connectivity. Overall design: Leaves 7 and 8 from three 4-week old Arabidopsis thaliana plants were collected from Col-0, 35S:ChEC127-GFP, and 35S:ChEC132-GFP plants for RNA-sequencing. Each samples contains 6 leaves and there are 3 bioreplicate for each genotype.