Cell-in-cell interaction between Rhizopus microsporus and Ralstonia pickettii

Endosymbioses represent complex and dynamic relationships between organisms that may involve pathogenic phases during their emergence. Here, we induce cell-in-cell interactions between the free-living Ralstonia pickettii and an endosymbiont-free strain of Rhizopus microsporus using fluidic force microscopy (FluidFM) to investigate the unstable early phase of endosymbiosis. The intracellular presence of R. pickettii initially compromised host fitness and triggered immune responses. Despite these effects, successful vertical transmission of bacteria across fungal generations enabled adaptation of the merged system through positive selection. High-throughput imaging revealed that the passaged system accommodated higher bacterial loads inside viable spores, albeit at the cost of reduced fungal growth. Comparative transcriptomic profiles between early and late propagation stages indicated adaptive resilience and resembled a shift from pathogenic antagonism to commensalism. This transition was characterized by attenuated expression of genes involved in cell wall remodeling and recalibrated reactive oxygen metabolism, both hallmarks of fungal defense. Our experimental system provides insights into the early processes of endosymbiosis, highlights the dynamics between pathogenic response and commensal coexistence, and supports the hypothesis that facultative pathogens can serve as evolutionary intermediates toward stable endosymbiotic interactions.