Reverse engineering high-level resistance to Bt Cry1Ac toxin in Plutella xylostella reveals a hormonal regulatory feedback pathway

Decoding the molecular mechanisms of insect resistance to Bacillus thuringiensis (Bt) toxins is crucial for the sustainable utilization of Bt-based bioinsecticides and transgenic crops. Our previous studies showed that a hormone-responsive transcription factor FOXO binds to an inserted short interspersed nuclear element (SINE, named SE2), causing MAP4K4 overexpression and resistance to Bt Cry1Ac toxin in Plutella xylostella. Furthermore, titers of two upstream signaling hormones (20-hydroxyecdysone and juvenile hormone) were also found to be elevated in the resistant strain, but it was unclear whether this was due to natural variation or a feedback pathway. Here, we established a homozygous knock-in strain (SE2-KI) using a reverse genetic approach to insert the SE2 retrotransposon into the MAP4K4 promoter of a Cry1Ac-susceptible strain. The SE2 insertion induced MAP4K4 overexpression, which in turn caused a downregulation of midgut receptors and an identical resistance phenotype to that seen in the evolved resistant strain. Moreover, SE2 insertion significantly increased the levels of two insect hormones providing definitive evidence for a positive feedback regulatory pathway. This study unveils an as yet uncharacterized hormonal regulatory feedback pathway orchestrating Cry1Ac resistance in P. xylostella, providing new insights into the molecular basis of Bt resistance and informing suitable field resistance management strategies.