BiFC for protein–protein interactions and protein topology: Discussing an integrative approach for an old technique

BiFC (Bi molecular F luorescence C omplementation) is one of the most widely used techniques to study protein–protein interactions as well as protein topology in living cells. This method allows the visualization of protein interactions or the analysis of their topology in the cell compartments where the proteins belong, without changing their chemical properties, as often occurs after mixing the content of different cellular compartments in cell extracts. Several laboratories use this method because it is relatively easy to perform; however, sometimes a positive protein–protein interaction BiFC signal (i.e., reconstitution of fluorescence of interacting protein pairs) does not necessarily mean that the tested proteins are actually interacting in vivo in a specific way. Here we describe the BiFC approach for assessing protein–protein interactions and for establishing protein topology and we discuss how to best perform this method to avoid false positive results when studying protein interactions in plant cells.