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.