Plasmodesmata (PD) are dynamic cell wall microchannels that facilitate the intercellular trafficking of RNA and protein macromolecules playing cell nonautonomous roles in the orchestration of plant development, growth, and plant defense. The trafficking of macromolecules and organelles within cells depends on cytoskeletal components and their associated motor proteins. Plant viruses evolved to hijack this transport system to move their infectious genomes to PD. Current efforts concentrate on dissecting the role of specific myosin motors in transporting plant or viral proteins to the channels. Here we describe a method that addresses the role of specific myosins by expression of myosin tails that cause the repression of myosin activity in a dominant-negative manner. As an example, we explain the use of myosin tails from Nicotiana benthamiana to address the role of N. benthamiana myosins in the targeting of PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) to PD.