ZEITLUPE in the Roots of Wild Tobacco Regulates Jasmonate-Mediated Nicotine Biosynthesis and Resistance to a Generalist Herbivore

The jasmonate (JA) phytohormone signaling system is an important mediator of plant defense against herbivores. Plants deficient in JA signaling are more susceptible to herbivory as a result of deficiencies in defensive trait expression. Recent studies have implicated the circadian clock in regulating JA-mediated defenses, but the molecular mechanisms linking the clock to JA signaling are unclear. Here, we report that wild tobacco (Nicotiana attenuata) plants rendered deficient in the clock component ZEITLUPE (ZTL) by RNA interference have attenuated resistance to the generalist herbivore Spodoptera littoralis. This effect can be attributed in part to reduced concentrations of nicotine, an abundant JA-regulated toxin produced in N. attenuata roots and transported to shoots. RNA interference targeting ZTL dramatically affects the root circadian clock and reduces the expression of nicotine biosynthetic genes. Protein-protein interaction experiments demonstrate that ZTL regulates JA signaling by directly interacting with JASMONATE ZIM domain (JAZ) proteins in a CORONATINE-INSENSITIVE1- and jasmonoyl-isoleucine conjugate-independent manner, thereby regulating a JAZ-MYC2 module that is required for nicotine biosynthesis. Our study reveals new functions for ZTL and proposes a mechanism by which a clock component directly influences JA signaling to regulate plant defense against herbivory.