Cytochrome P450 enzymes in the Arabidopsis indole-3-acetonitrile metabolic network: Biological functions and protein-protein interactions

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As a model for induced chemical defences, the network of indole-3-acetonitrile (IAN) metabolism and camalexin biosynthesis is analysed in Arabidopsis. A set of multiple mutants in biosynthetic genes is generated to evaluate the roles of different subclasses of tryptophan-metabolites in pathogen resistance. Here, the deletion of an entire cluster of 16 cytochrome P450-encoding genes was achieved somatically following a TALE-nuclease-based approach and now stable deletion lines are aimed for. In an untargeted Co-IP approach, physical interaction of the camalexin biosynthetic enzyme CYP71B15 (PAD3) with other P450-enzymes involved in IAN metabolism, as well as with regulatory proteins, was observed. We will now systematically analyse functional protein-protein interactions by targeted Co-IP, Förster resonance energy transfer, yeast-2-hybrid analysis, and functional coexpression in yeast and Nicotiana benthamiana, to gain insight into the formation of metabolons involved in metabolic defence.