Metabolic engineering of Barbarea vulgaris identifies β-amyrin synthase LUP5 as a determinant of saponin-mediated insect resistance

Plant–insect coevolution drives the diversification of specialized metabolites, yet the genetic basis of ecotypic variation in defensive chemistry remains poorly understood. The wild crucifer Barbarea vulgaris comprises two ecotypes—an insect-resistant G-type and a susceptible P-type—characterized by distinct triterpenoid saponin profiles.

To investigate the causal role of saponin composition in insect resistance, we developed a stable transformation system for B. vulgaris . We expressed the G-type β-amyrin synthase gene ( LUP5 ) in both ecotypes and silenced the cytochrome P450 gene CYP72A552 in the G-type. We combined metabolic profiling with insect feeding assays to assess functional outcomes.

Ectopic expression of G-type LUP5 in the susceptible P-type conferred up to 95% reduction in Plutella xylostella feeding, accompanied by increased accumulation of three hederagenin-derived monodesmosidic saponins. In contrast, silencing of CYP72A552 reduced hederagenin derived saponin levels but did not affect herbivory, suggesting threshold-dependent resistance.

We provide direct in planta evidence that LUP5 is a key determinant of ecotypic insect resistance in B. vulgaris . Our findings establish a functional link between oxidosqualene cyclase activity, saponin structural diversification, and ecological defense, highlighting the roles of both sapogenin backbone and glycosylation pattern in saponin-mediated herbivore deterrence.