MYC2 and MYC3 orchestrate pith lignification to defend Nicotiana attenuata stems against a stem-boring weevil.

Lignin is a key structural polymer that also serves as a potent defense against biotic stress. Herbivore-induced, jasmonate-dependent pith lignification in Nicotiana attenuata plays a crucial role in defense against the stem-borer Trichobaris mucorea. However, the regulatory mechanisms underlying herbivore-induced lignification remain largely unknown. We demonstrate that NaMYC2 and NaMYC3 orchestrate pith-specific lignification in response to T. mucorea attack. RNA-seq analysis reveals that monolignol biosynthetic genes and polymerization-associated genes fail to be induced in Namyc2/3 double mutants upon T. mucorea attack. Among NaMYC2/3-dependent genes in the attacked pith, we identify NaTHT1, responsible for synthesizing the noncanonical monolignol N-FT. Using Natht1 mutants, we further show that N-FT plays a key role in stem defense. Additionally, we identify NaNEC1a and NaNEC1c, NaMYC2/3-dependent superoxide dismutases in the pith. Nanec1a/1c double mutants exhibit reduced lignification and enhanced larval performance, supporting a direct link between superoxide metabolism and induced lignification. Our findings reveal a NaMYC2/3-mediated regulatory network in the pith that integrates monolignol biosynthesis, lignin polymerization, and noncanonical monolignol biosynthesis, thereby enhancing stem defense against T. mucorea.