Elucidation of the biological function of an 83 kb large cytochrome P450 gene cluster in Arabidopsis

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Characteristic for plants is the presence of clusters of duplicated genes. Often these genes are functionally redundant, which makes it difficult or impossible to draw conclusions on biological functions from the phenotypic analysis of single insertion mutants. Recently, genome editing has been established for plants, which also allows mutation of more than one gene. However, targeted deletion of larger gene clusters is still challenging. As a proof of concept study, we have been focusing on the largest cluster of duplicated cytochrome P450 genes (belonging to the CYP71B subfamily) in Arabidopsis thaliana, which spans 83 kb. By a CRISPR/Cas9-based approach we have generated homozygous mutants in which this cluster is deleted. These plants are fully vital, but show a modified composition of defence-related metabolites. To specifically identify in vivo functions of the cluster genes, also partial deletion and complementation lines will be generated and analysed by comparative metabolomics in different tissues and in response to pathogens and abiotic stresses. This will be complemented with the characterisation of the respective P450 enzymes expressed in yeast and Nicotiana benthamiana. We will then generate combinations with deletion and insertion mutations in other CYP71B genes to enhance our functional understanding of this large diversified subfamily.