Isolation and transfection of plant protoplasts for studying infection and replication of plant viruses.

The development of single-cell protoplast systems is considered a breakthrough in the history of plant virology. The protoplast system has several advantages over the use of whole plants, 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. Thus, the single-cell protoplast system offers unprecedented opportunities in dissecting viral molecular processes at the cellular level such as translation, replication, movement, virion assembly and RNA recombination. With the availability of global gene expression profiling technologies, namely microarrays, the protoplast system permits the spatial and temporal analysis of global gene responses to viral infections, which is essential for decoding early signalling pathways associated with compatible and incompatible infections. In this chapter, we will summarize the documented technology for protoplast isolation and transfection, and applications of plant protoplasts to the molecular characterization of viral infection and replication processes as well as virus-plant interactions. As a case study, we will provide a detailed protocol for the isolation and transfection of Arabidopsis thaliana protoplasts.