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.