Functional analysis of tobacco PPO genes in insect resistance

Polyphenol oxidase (PPO) is a copper-containing metalloenzyme that catalyzes the oxidation of polyphenols to quinones and is implicated in plant metabolism, enzymatic browning, and stress adaptation. Despite extensive study of PPOs, the isoform-specific contributions of tobacco PPOs to herbivore defense have not been functionally resolved. Here, we systematically analyzed the tobacco PPO gene family and identified 15 PPO members. Among these, NtPPO1, NtPPO2, NtPPO3, NtPPO4, and NtPPO11 were strongly and consistently induced by insect feeding. We then functionally dissected these insect-responsive NtPPOs using complementary loss- and gain-of-function approaches. CRISPR/Cas9 editing generated NtPPO2 NtPPO3 NtPPO4 and NtPPO2 NtPPO3 NtPPO11 triple mutants, as well as an NtPPO1 NtPPO2 NtPPO3 NtPPO4 NtPPO11 quintuple mutant; all mutants showed a pronounced decrease in PPO activity, a significant increase in total phenolic content, and markedly reduced resistance to Spodoptera litura. In contrast, transgenic lines individually overexpressing NtPPO2, NtPPO3, or NtPPO4 exhibited substantially elevated PPO activity, lowered total phenolic levels, and enhanced resistance to S. litura. Collectively, these results provide direct genetic and biochemical evidence that insect-inducible NtPPOs act as positive regulators of tobacco anti-herbivore defense, likely through modulating phenolic oxidation and quinone-mediated toxicity. This work establishes isoform-specific functions of tobacco PPOs in plant–insect interactions and identifies promising targets for improving insect resistance in Solanaceae crops.