Abstract
Effective rhizobium-legume symbiosis depends on multiple molecular signaling pathways, integrating not only classical nodulation factors and surface polysaccharides but also diverse protein secretion systems. Among them, the Type VI Secretion System (T6SS) has emerged as a key player, due to its dual roles in interbacterial competition and interactions with eukaryotic hosts, though its contribution to symbiosis remains unclear. Key regulatory messengers, including the main autoinducer of the quorum sensing (QS) systems, the N-acyl homoserine lactones (AHLs), and the second messenger cyclic di-GMP (c-di-GMP), modulate the transition between motility and biofilm formation, especially in the context of bacteria interacting with eukaryotes, including rhizobia. While c-di-GMP's impact on exopolysaccharide production in these organisms is well established, its influence on protein secretion systems, particularly in conjunction with QS, is largely unexplored. To contribute to the study of such interplay, we artificially increased intracellular c-di-GMP levels by overexpressing a heterologous diguanylate cyclase in three Sinorhizobium fredii strains of agronomic relevance. This engineering revealed strain-specific outcomes, since elevated c-di-GMP enhanced biofilm development in two strains, but reduced it in another. Furthermore, using β-galactosidase expression assays, we confirmed that both high c-di-GMP and/or AHL concentrations contribute to the transcriptional activation of T6SS. These results demonstrate a direct regulatory link between c-di-GMP, QS signals, and T6SS expression, shedding light on the multilayered control mechanisms that structure beneficial rhizobia-plant interactions.