Chewing insect-induced activation of NtNAC29 and NtNAC94 enhances plant defense via NtCPI8 in tobacco

The cotton bollworm Helicoverpa armigera ( Lepidoptera : Noctuidae ) is a major pest of numerous crops. Plant responses to cotton bollworm attack are generally attributed to two primary factors: physical wounding and components of insect oral secretions. Discriminating between these responses is critical for understanding plant defense and developing pest control strategies. In this study, we found that cotton bollworm infestation specifically upregulated the expression of NtNAC29 and NtNAC94 , whereas mechanical wounding did not. Functional analyses revealed that silencing either gene promoted bollworm growth, whereas overexpression suppressed larval development. These results demonstrated that NtNAC29 and NtNAC94 had pivotal roles in enhancing plant resistance to bollworm. Further analyses showed that both NAC transcription factors regulated the expression of Cysteine Protease Inhibitor 8 ( NtCPI8 ) by binding to specific sites within the NtCPI8-1 and NtCPI8-2 promoters. Consistently, NtCPI8 exhibited a similar defensive role, significantly inhibiting cotton bollworm growth as reflected by reduced larval weight gain and shorter body length. Overall, our findings suggest that tobacco plants recognize cotton bollworm attack and activate downstream defense responses, including the induction of NtNAC29 and NtNAC94 . These transcription factors in turn upregulate NtCPI8 expression, thereby strengthening plant resistance against insect herbivory. Notably, this NAC-CPI regulatory module is conserved among different crop species, providing a promising target for improving crop protection against herbivorous insects.