Replication Assay of Begomovirus in Arabidopsis Protoplasts

Protoplasts are isolated plant cells from which the cell walls have been removed by treatment with fungal cellulase and macerozyme enzymes, which degrade the primary components of the cell wall. The protoplasts are totipotent, sensitive, and versatile; thereby, they have been extensively used to study signal transduction pathways, cell-autonomous responses, and replication of plant viruses. This system has several advantages over the use of whole plants for viral replication, including a high percentage of infected cells and uncoupling virus movement from replication assays. Here, we describe a simple and efficient protocol for begomovirus transfection and replication in Arabidopsis protoplasts. The method consists of four steps, (i) protoplast isolation, (ii) PEG-calcium transfection of begomovirus infectious clones, (iii) elimination of input plasmid DNA by DpnI digestion, and (iv) quantification of the viral newly synthesized DNA by qPCR. The protoplasts can be transformed efficiently with begomovirus infectious clones, and virus replication can be monitored by the accumulation of nascent viral DNA in the infected protoplasts.