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

Plant-insect coevolution has been a major driver of specialized metabolite diversification, yet the genetic basis of natural 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 relationship between saponin composition and insect resistance, we established a stable transformation system for B. vulgaris. Expression of the G-type {beta}-amyrin synthase gene LUP5 in the susceptible P-type conferred up to a 95% reduction in Plutella xylostella feeding, accompanied by increased accumulation of three hederagenin-derived monodesmosidic saponins. Comparison of LUP5 expression driven by its native promoter and by the constitutive 35S promoter revealed that the native promoter leads to increased hederagenin accumulation and is activated later in development, which may prevent early metabolic stress and allow coordinated expression of downstream pathway genes. In contrast, silencing of CYP72A552 by RNAi decreased total hederagenin levels by approximately 40% without affecting resistance, indicating threshold-dependent defense. Our results provide direct in planta evidence that LUP5 is a key determinant of natural variation in insect resistance in B. vulgaris, underscoring the pivotal role of the saponin backbone in herbivore deterrence. By linking promoter activity to metabolite structural diversity, this work provides mechanistic and conceptual insight into how plants coordinate specialized metabolism and defense.