This fully updated book aims to facilitate the study of the nanochannels that connect plant cells, known as plasmodesmata, and to instigate new research that will further advance our knowledge of these structures. Beginning with the general structural composition and regulation of plasmodesmata as well as their role in plant development and disease, the volume continues with chapters exploring plasmodesmata architectures and distribution in cell interfaces, approaches to dissect plasmodesmata composition, protocols to quantify changes in plasmodesmata permeability using fluorescent tracers and mobile proteins, as well as a section with protocols that contribute to plasmodesmata research but fall outside the previous classifications. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Plasmodesmata: Methods and Protocols, Second Edition serves as a vital guide for all plant scientists, both novice and expert, especially those studying the intricacies of cell-to-cell communication pathways.