Mutations in CYP94B3 Genes Enhance Insect Resistance in Tobacco

JA-Ile has been recognized as a crucial signaling molecule in plant defense responses against insect herbivory. The oxidative metabolism catalyzed by cytochrome P450 monooxygenase CYP94B3 is considered one of the major pathways regulating JA-Ile catabolism. In this study, four CYP94B3 homologs were identified in cultivated tobacco (Nicotiana tabacum), two of which were found to be mutated, suggesting possible functional redundancy among these genes. To determine whether mutation of CYP94B3 could enhance insect resistance in tobacco, we generated knockout lines using the CRISPR-Cas9 system. The results showed that homozygous CYP94B3 knockout lines exhibited significantly enhanced insect resistance. In addition, the JA-Ile content was elevated in the knockout lines, and the levels of insect resistance-related secondary metabolites, including TPI and nicotine, were also significantly increased, which may account for the enhanced insect resistance. Taken together, these findings provide a basis for the genetic improvement of insect resistance in plants through targeted manipulation of the JA-Ile metabolic pathway.