Visualization and Quantification of Cell-to-cell Movement of Proteins in Nicotiana benthamiana.

Cell-to-cell movement of proteins through plasmodesmata is a widely-established mechanism for intercellular signaling in plants. Current techniques to study intercellular protein translocation rely on single-cell transformation using particle bombardment or transgenic lines expressing photo-inducible fluorophores. The method presented here allows visualization and objective quantification of (effector) protein movement between N. benthamiana leaf cells. Agroinfiltration is performed using a single binary vector encoding a GFP-tagged protein of interest that is either mobile or non-mobile (MP; non-MP), together with an ER-anchored mCherry. Upon creation of mosaic-like transformation patterns, cell-to-cell movement of the MP can be followed by monitoring translocation of the GFP signal from mCherry labeled transformed cells into neighboring non-transformed cells. This process can be visualized using confocal microscopy and quantified following protoplast isolation and flow cytometric cell analysis. This method overcomes the limitations of existing methods as it allows rapid and objective quantification of protein translocation without the need of creating transgenic plants.