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

Transient expression assays are invaluable complements to the stable transgenic assay for studying gene functions by providing desirable time and labor efficiencies and high-throughput potential or circumventing 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 PCR amplicon-mediated protoplast transient (PROMPT) assay by using overlapping PCR assembled gene expression cassettes for Arabidopsis protoplast transfection without 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.