Multi-omics insights reveal a divergent type III secretion system-associated secretome in plant growth-promoting rhizobacterial Pseudomonas fluorescens.

The type III secretion system (T3SS) is a well-characterized virulence apparatus in pathogenic bacteria; however, its role in beneficial rhizobacteria remains poorly defined. In the current study, we integrated multi-omics analyses with molecular genetics to systematically characterize the regulatory network and secretory output of the T3SS in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens 2P24. The canonical effector repertoire of 2P24 is markedly streamlined, and the conserved effector homolog HopM12P24 has lost virulence-associated activities. The sigma factor RspL is a root-responsive regulator that reprograms 31.92% of detected bacterial genes, promoting energy generation and metabolic pathways while repressing behavioral and signaling programs. Furthermore, T3SS integrity directly governs extracellular protein allocation by facilitating the secretion of non-canonical metabolic enzymes-including a validated hydrolase and decarboxylase-while restricting the release of flagellar components. Collectively, these findings establish the RspL-T3SS axis as an integrated regulatory and secretory module that coordinates transcriptional reprogramming with selective protein secretion during root interaction. This study expands our understanding of the classic virulence-associated T3SS as an adaptive machinery for rhizosphere colonization and yields mechanistic insights into bacterial rhizosphere fitness.