Engineering Pseudomonas protegens as a Targeted Antifungal-Effector Delivery Chassis via Heterologous Type III Secretion System Reconstitution

Abstract

This study addresses the core challenge of Fusarium wilt control in agricultural production. We successfully reconstituted a functional heterologous type III secretion system (T3SS) from Photorhabdus luminescens in the biocontrol bacterium Pseudomonas protegens Pf-5, creating an engineered molecular syringe for targeted delivery of antifungal effectors. The system is activated under low-calcium conditions, achieved by cultivation in calcium-limited medium followed by EGTA-mediated chelation of residual Ca²⁺, enabling conditional secretion of effector proteins. By fusing the antifungal protein Bg9562 to the N-terminal secretion signal of the T3SS effector LopT and co-expressing it with the cognate chaperone SlcT, we obtained fluorescence-based evidence for T3SS-dependent delivery of Bg9562 into the hyphae of multiple Fusarium species. The engineered strain exhibited enhanced rhizosphere colonization, promoted plant growth and conferred improved protection against tomato Fusarium wilt, restoring plant height to levels approaching healthy controls. We further demonstrated the modularity of this platform by successfully transferring it into Pseudomonas koreensis D26, a strain known for its plant growth-promoting properties, indicating broad applicability across biocontrol-relevant pseudomonads. This work establishes a versatile T3SS-based delivery platform for precision biocontrol, offering a generalizable strategy for engineering beneficial rhizobacteria.