Auxin‐Induced Nicotine Inhibition Is Mediated by <scp>NaARF5</scp> Through the Suppression of <i>NaERF1‐Like</i> Expression and Interaction With <scp>NaERF1‐Like</scp> in <scp> <i>Nicotiana attenuata</i> </scp>

Nicotine is the most abundant alkaloid produced by Nicotiana species against insect herbivores. Although it has been known for decades that auxin can strongly inhibit nicotine biosynthesis, the underlying mechanisms have remained unclear. Here we demonstrated that NaERF1-like, the orthologue of NtERF199, is a key regulator of nicotine biosynthesis through gene-editing and stable overexpression. NaERF1-like expression was strongly inhibited by auxin treatments, suggesting that it is a target gene regulated by auxin. Yeast two-hybrid screening revealed that NaARF5, an auxin response factor, interacts with NaERF1-like. This interaction was further confirmed by GST pull-down, bimolecular fluorescence complementation, and split-luciferase complementation. NaARF5 acts as a key negative regulator of nicotine production since both the expression of nicotine-related genes and nicotine production increased significantly in NaARF5 mutants generated by CRISPR/Cas9 but was severely impaired in NaARF5 overexpression lines. Notably, the inhibition of nicotine-related genes by auxin, including NaERF1-like, is alleviated in NaARF5 mutants. Furthermore, the interaction of NaARF5 with NaERF1-like reduced the binding and activating ability of NaERF1-like to the promoter of NaPMT1.1, a key enzyme gene involved in nicotine biosynthesis, as well as the transcriptional activity of NaQPT2. Thus, we uncovered a new auxin-mediated regulatory pathway for the inhibition of nicotine biosynthesis through NaARF5 by targeting NaERF1-like. Our findings provide novel insights into the molecular mechanisms underlying the inhibition of defensive metabolites by auxin and establish NaARF5 as a valuable breeding target for manipulating nicotine production.