Induced endosymbiosis between a fungus and bacterium reveals a shift from antagonism to commensalism

Endosymbioses represent dynamic relationships between organisms that may involve antagonistic phases during their emergence. Here, we induced cell-in-cell interactions between the free-living bacterium Ralstonia pickettii and an endosymbiont-free strain of the fungus Rhizopus microsporus using fluidic force microscopy to investigate the early phase of endosymbiosis formation. Following the implantation of bacteria into the cytosol, the rapid proliferation of R. pickettii compromised host fitness, as evidenced by reduced fungal viability, and triggered immune responses characterized by upregulated expression of stress-related defense genes. Vertical transmission of bacteria across fungal generations enabled repeated rounds of selective passaging, ultimately resulting in transcriptional relaxation of the fungal defense response. High-throughput-imaging revealed that the propagated system accommodated higher bacterial loads within viable spores, with a corresponding reduction in fungal growth. The observed physiological changes and comparative fungal transcriptomic profiles indicated adaptive resilience and a shift from antagonism to commensalism. This transition was characterized by attenuated expression of genes involved in cell wall remodeling and reactive oxygen metabolism. Our experimental system provides insights into the early processes of endosymbiosis, supporting the hypothesis that facultative intracellular pathogens can serve as intermediates toward stable endosymbiotic relationships.