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Summary  Protein networks and signaling cascades are key mechanisms for intra‐ and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The bimolecular fluorescence complementation (BiFC) assay has become a routine tool for  in vivo  analysis of protein–protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for  in planta  analysis are still subject to very low signal‐to‐noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive  in planta  analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus ( mV enus) split at residue 210, and contains an integrated  mT urquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined  d ouble  O RF  e xpression ( pDOE ) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused  mV enus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin‐like (CML) protein, and by showing that heterotrimeric G‐protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1  in planta , and that mutation of the so‐called PLDα1 ‘DRY’ motif abolishes both of these interactions.

Significant reduction of Bi FC non‐specific assembly facilitates in planta assessment of heterotrimeric G‐protein interactors