Identification and characterization of Arabidopsis thaliana transgenic strains expressing artificial microRNAs that are defective in response to indole-3-carbinol

Indole-3-carbinol (I3C) is a small molecule produced in the Cruciferae family following mechanical damage or insect attack, as a breakdown product of glucosinolates. I3C functions in cruciferous vegetables as a protective agent against foraging. It is also implicated in human health with significant anti-cancer properties. In Arabidopsis, exogenously applied I3C inhibits root elongation in a dose-dependent manner, as it acts as an auxin antagonist which competes for the binding site of the auxin receptor TIR1. Yet, little else is known about the I3C signaling pathway in plants. This work aims at identifying novel components of the pathways interacting with I3C signaling in Arabidopsis. We employed a genome-scale artificial microRNA screen, using modified Arabidopsis thaliana miR319a precursor to express artificial miRNAs (amiRNAs) targeting mRNA sequences of protein kinase families, and identified two strains with either enhanced or reduced responses to exogenous I3C. This screening method overcomes gene redundancy, enabling the identification of novel protein. We identified wall-associated kinases acting as negative regulators of I3C action and phosphatidylinositol phosphate kinases as positive mediators of I3C signaling. These kinases intersect I3C signaling in auxin-independent pathways.