Fha initiates the inside-out assembly of the type VI secretion system.

How cells orchestrate the quantity and location while building complex intracellular machinery remains a fundamental biological question. The type VI secretion system (T6SS) is a powerful bacterial apparatus that delivers toxins into a variety of target cells, yet its assembly process is not fully understood. Contrary to the prevailing "outside-in" model, our study reveals a novel "inside-out" assembly mechanism in Acidovorax citrulli, an important plant pathogen. Fha dictates the formation of the inner-membrane complex and the baseplate and directly interacts with these key components. Additionally, Fha undergoes liquid-liquid phase separation (LLPS), forming condensates that selectively recruit essential T6SS proteins. The Fha homolog in the human pathogen Pseudomonas aeruginosa exhibits conserved functions in condensate formation. These findings unveil an inside-out model for T6SS assembly primed by an LLPS-mediator, Fha, that is crucial for promoting assembly efficiency.