Isolation and Transfection of Plant Mesophyll Protoplasts for Virology Research

Protoplasts are the naked plant cells lacking the rigid cell wall and have been broadly utilized as an excellent tool to study the molecular virus-plant interactions, particularly at the early stages of the infection process, such as virion disassembly, viral genome translation, intracellular trafficking, and virus replication. Compared to the use of whole plants, the protoplast system has several major advantages in plant virology research, including homogeneous cell populations, high percentage of infected cells, synchronous infection, effects free from other cells/tissues, and ease of extraction of the viral RNA. This chapter describes a simple, streamlined, and efficient protocol for isolation and purification of mesophyll protoplasts from the model plants Arabidopsis thaliana and Nicotiana benthamiana, and subsequent transfection of the isolated protoplasts with a potyvirus infectious clone.