Engineering and Functional Expression of the Type III Secretion System in Xenorhabdus: Enhancing Insecticidal Efficacy and Expanding T3SE Libraries

Entomopathogenic nematode symbiotic bacteria (EPNB) enhance nematode insecticidal capacity through symbiosis. This study cloned the complete 32-kb type III secretion system (T3SS) gene cluster from Photorhabdus luminescens TT01 using Red/ET recombineering and functionally expressed it in T3SS-deficient Xenorhabdus stockiae HN_xs01. Heterologous T3SS expression significantly enhanced HN_xs01 adhesion and invasion capabilities in CF-203 cells. In Helicoverpa armigera models, the engineered strain induced severe intestinal damage by suppressing antimicrobial peptide expression and demonstrated improved colonization and biocontrol efficacy (LC₅₀ decreased by 3.7-fold). Crucially, the TT01 derived T3SS mediated delivery of XopA─a novel effector exhibiting YopJ-family homology and characteristic T3SS effector features─into host cells. These findings establish the synthetic biology-driven potential of T3SS and its effectors for biological control applications while providing a mechanistic framework for future research.