Artificial symbiont replacement in a vertically-transmitted plant-bacterium association provides insights into the basis for specificity

Some plants engage in permanent, vertically transmitted symbioses with bacteria. Often, these bacteria are hosted extracellularly in specific structures on the leaves, where they produce specialized bioactive metabolites that benefit their host. These associations are highly specific, with one plant species associating with a single bacterial species, but little is known about how these symbioses originate and how specificity is maintained. In this study, we show that the symbiotic association between a wild yam and a bacterium can be manipulated experimentally, and that bacteria-free plants are open to colonization by environmental bacteria. Through metabolic profiling, we show that the endophytic niche is rich in organic acids and intermediates of the TCA cycle. Environmental bacteria capable of utilizing these acids, such as the soil bacterium Pseudomonas putida, readily colonize aposymbiotic plants. However, successful colonization is contingent upon the absence of the vertically-transmitted symbiont and an active type VI secretion system. Unexpectedly for a vertically-transmitted symbiosis, these findings suggest that microbe-microbe interactions, including antagonism, may play a crucial role in maintaining the specificity of an association. However, low transmission rates of synthetic symbionts provide evidence that transmission barriers or bottlenecks may still occur, further enforcing partner fidelity. Together, these results highlight the complexity of mechanisms underlying mutualistic associations, and provide insights into the evolution of bacterial leaf symbiosis.