Updated project metadata. Arabidopsis thaliana efficiently synthesizes the antifungal phytoalexin camalexin without apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting channeling of the biosynthetic pathway involving an enzyme complex. To identify such protein interactions, two independent untargeted co-immunoprecipitation (Co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits were performed and the camalexin biosynthetic P450 enzymes were co-purified. These interactions were confirmed by targeted Co-IP and förster resonance energy transfer measurements based on fluorescence lifetime imaging (FLIM-FRET). Furthermore, interaction of CYP71A13 and Arabidopsis P450 reductase 1 (ATR1) and recruitment of cytosolic gamma-glutamyl peptidase 1 (GGP1) to the endoplasmic reticulum (ER) was observed. An increased substrate affinity of CYP79B2 in presence of CYP71A13 was shown, indicating substrate channeling by the complex. During camalexin biosynthesis, indol-3-acetonitrile (IAN) is activated by CYP71A13 and glutathionylated to GS-IAN. To clarify whether this is mediated by a specific glutathione transferase (GST), CYP71A13 was expressed together with each of the 54 Arabidopsis GSTs in S. cerevisiae and GS-IAN-synthesis was observed for GSTU2. Corresponding studies of knock-out and overexpression plants did not show an altered camalexin content. However, an influence of the closely related GSTU4 on camalexin biosynthesis was demonstrated. GSTU4 is co-expressed with tryptophan- and camalexin specific enzymes and physically interacts with CYP71A13. Surprisingly, camalexin concentrations were reduced in knock-out and elevated in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a regulatory role or is involved in transport or a competing mechanism.