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

作为诱导化学防御的模型，分析了拟南芥中吲哚-3-乙腈(Ian)代谢和Camalexin生物合成的网络。产生了一组生物合成基因的多个突变体，以评估不同亚类色氨酸代谢物在病原菌抗性中的作用。在这里，16个细胞色素P450编码基因的整个簇的缺失是通过基于故事核酸酶的方法在体细胞上实现的，现在目标是稳定的缺失线。在非靶向的Co-IP方法中，观察到Camalexin生物合成酶CyP71B15(PAD3)与参与Ian代谢的其他P450酶以及调节蛋白的物理相互作用。我们现在将通过有针对性的Co-IP、Förster共振能量转移、酵母-2-杂交分析以及在酵母和本氏烟草中的功能共表达来系统地分析蛋白质之间的功能相互作用，以深入了解参与代谢防御的代谢物的形成。

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.