A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies.

Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are prone to self-assembly independent of a protein-protein interaction event. To circumvent this problem, several modifications of the technique have been suggested, but these modifications have not lead to improvements in plant BiFC protocols. Therefore, it remains crucial to include appropriate internal controls. Our literature survey of recent BiFC studies in plants shows that most studies use inappropriate controls, and a qualitative rather than quantitative read-out of fluorescence. Therefore, we provide a cautionary note and beginner's guideline for the setup of BiFC experiments, discussing each step of the protocol, including vector choice, plant expression systems, negative controls, and signal detection. In addition, we present our experience with BiFC with respect to self-assembly, peptide linkers, and incubation temperature. With this note, we aim to provide a guideline that will improve the quality of plant BiFC experiments.