Enhanced protoplast assay by transfecting PCR-assembled gene expression cassettes with telomeric repeats and thiophosphate modifications

Abstract Transient expression assays are invaluable complements to the stable transgenic assay for studying gene functions, because they possess desirable time, labor efficiencies and high-throughput potential or circumvent technical difficulties of stable transgenic expression. The protoplast transient expression system is one of the mainstream transient expression assays used in plant research. Here, we developed a P C R amplic o n- m ediated p rotoplast t ransient (PROMPT) assay by using overlapping PCR assembled gene expression cassettes for Arabidopsis protoplast transfection instead of plasmid DNA, thereby bypassing the need for time- and labor-consuming plasmid construction. When 200 μl of Arabidopsis protoplasts were transfected with 1 μg of PCR amplicons or plasmid DNA, we detected substantially higher gene expression in the former. Moreover, we found that adding telomeric repeats and thiophosphate modifications to the 5’ end of the nonsense strand through the reverse primer could further increase the PCR amplicon-mediated gene expression in protoplasts. Importantly, these improvements could also be applied to the protoplast assays in other dicot and monocot species including tobacco, rice and wheat. In addition, the subcellular localization of immune receptor FLS2 could be analyzed by PROMPT method. The PROMPT assay allows an accelerated and robust transient gene expression in protoplasts from diverse plant species.