A new cyanogenic metabolite in Arabidopsis required for inducible pathogen defense

Thousands of putative biosynthetic genes in Arabidopsis thaliana have no known function, suggesting that there are numerous molecules contributing to plant fitness that have not yet been discovered(1,2). Prime among these uncharacterized genes are cytochromes P450 upregulated in response to pathogens(3,4). Starting with a single pathogen-induced P450(5), CYP82C2, we used a combination of untargeted metabolomics and co-expression analysis to uncover the complete biosynthetic pathway to a previously unknown Arabidopsis metabolite, 4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN), which harbors cyanogenic functionality that is unprecedented in plants and exceedingly rare in nature(6,7). The aryl cyanohydrin intermediate in the 4-OH-ICN pathway reveals a latent capacity for cyanogenic glucoside biosynthesis(8,9) in Arabidopsis. By expressing 4-OH-ICN biosynthetic enzymes in Saccharomyces cerevisiae and Nicotiana benthamiana, we reconstitute the complete pathway in vitro and in vivo and validate the functions of its enzymes. 4-OH-ICN pathway mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with a role in inducible pathogen defense. Arabidopsis has been the preeminent model system(10,11) for studying the role of small molecules in plant innate immunity(12); our results uncover a new branch of indole metabolism distinct from the canonical camalexin pathway, and support a role for this pathway in the Arabidopsis defense response.(13) These results establish a more complete framework for understanding how the model plant Arabidopsis uses small molecules in pathogen defense.