Recombinant humoral immune proteins differentially modulated Cry1Ac susceptibility in Plutella xylostella

The insect immune system is closely associated with Bt resistance. Our previous studies have shown that silencing humoral immune genes (PxPGRP2, PxIMD, PxSPN1 and PxPPO1) in Plutella xylostella significantly affected larval susceptibility to Cry1Ac and also impacts fecundity. However, the functional roles of these humoral immunity genes at the protein level remain unclear. In the present study, we obtained the corresponding recombinant proteins through a prokaryotic expression, evaluated their effects on Cry1Ac susceptibility in P. xylostella, and examined changes in biological traits and transcription levels of upstream and downstream genes in the absence of Cry1Ac treatment.The results showed that pET41a-PxIMD reduced larval susceptibility of P. xylostella to Cry1Ac, accompanied by altered expression of IMD pathway-related genes and downregulation of Cry1Ac receptor expression. In contrast, pET41a-PxPGRP2 enhanced the toxicity of Cry1Ac to P. xylostella, accompanied by downregulation of IMD pathway-related genes. pET41a-PxSPN1 improved the insecticidal efficacy of Cry1Ac via suppression of prophenoloxidase-activating system (proPO-AS)-related gene expression and reduction of midgut phenoloxidase (PO) content, whereas pET41a-PxPPO1 treatment decreased larval susceptibility to Cry1Ac. In addition, all recombinant protein treatments resulted in fitness costs, including reduced fecundity. Collectively, these findings confirm the functional association between humoral immunity and Cry1Ac susceptibility in P. xylostella, and highlight the potential of pET41a-PxPGRP2 and pET41a-PxSPN1 recombinant proteins as synergists for Cry1Ac.