Quantitative Analysis of Protein‐Protein Interactions by Split Firefly Luciferase Complementation in Plant Protoplasts

Abstract

This unit describes the split firefly luciferase complementation (SFLC) assay, a high-throughput quantitative method that can be used to investigate protein-protein interactions (PPIs) in plant mesophyll protoplasts. In SFLC, the two proteins to be tested for interaction are expressed as chimeric proteins, each fused to a different half of firefly luciferase. If the proteins interact, a functional luciferase can be transitorily reconstituted, and is detected using the cell-permeable substrate D-luciferin. An advantage of the SFLC assay is that dynamic changes in PPIs in a cell can be detected in a near real-time manner. Another advantage is the unusually high DNA co-transfection and protein expression efficiencies that can be achieved in plant protoplasts, thereby enhancing the throughput of the method.

Targeted Transcriptional Activation in Plants Using a Potent Dead Cas9–Derived Synthetic Gene Activator

“…At least three biological replicates should be conducted to determine the optimal sgRNA. (C) A binary vector containing the expression cassettes of dCas9-TV and the optimal sgRNA is constructed for Agrobacterium-mediated transformation of Arabidopsis and rice (Oryza sativa ;Kramer & Coen, 2001), agarose gel electrophoresis (see Current Protocols article; Voytas, 2000), Sanger DNA sequencing (see Current Protocols article; Slatko, Albright, Tabor, & Ju, 1999), plasmid DNA extraction by maxiprep (see annotation to step 33), isolation of Arabidopsis mesophyll protoplasts (Yoo, Cho, & Sheen, 2007; see Current Protocols article; Li & Zhang, 2014) or rice leaf sheath protoplasts (Zhang et al, 2011b), electroporation (see Current Protocols article; Seidman, Struhl, Sheen, & Jessen, 2001), colony PCR (see Current Protocols article; Woodman, 2008), stable transformation of Arabidopsis (Clough & Bent, 1998) or rice (Hiei, Ohta, Komari, & Kumashiro, 1994), and transformant selection for Arabidopsis (Clough and Bent, 1998;Zhang, Henriques, Lin, Niu, & Chua, 2006) or rice (Hiei et al, 1994;Hiei & Komari, 2008) NOTE: Experiments involving PCR and RNA require extremely careful technique to prevent contamination and RNA degradation.…”