Eukaryote–eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms that act to stabilize emergent endosymbioses—between two fundamentally selfish biological organisms—are unclear. Theory suggests that enforcement mechanisms, which punish misbehavior, may act to stabilize such interactions by resolving conflict. However, how such mechanisms can emerge in a facultative endosymbiosis has yet to be explored. Here, we propose that endosymbiont–host RNA–RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model facultative endosymbiosis between Paramecium bursaria and Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived messenger RNA (mRNA) released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term “RNAi collisions,” represents a mechanism that can promote stability in a facultative eukaryote–eukaryote endosymbiosis. Specifically, by imposing a cost for breakdown of the endosymbiosis, endosymbiont–host RNA–RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions.

真核生物-真核生物内共生是叶绿体（质体）细胞器传播的原因。推动新细胞器建立的代谢和遗传整合需要稳定性，但稳定新兴的内共生的机制 - 在两个从根本上自私的生物有机体之间 - 尚不清楚。理论表明，惩罚不当行为的执行机制可能会通过解决冲突来稳定这种互动。然而，这些机制如何在兼性内共生中出现仍有待探索。在这里，我们提出内共生体-宿主 RNA-RNA 相互作用，由内共生体种群的消化引起，可能导致宿主生长的成本，以破坏内共生。使用草履虫间兼性内共生模型和小球藻spp.，我们证明这种机制依赖于宿主 RNA 干扰 (RNAi) 系统。我们通过小 RNA (sRNA) 测序揭示了内共生体消化后释放的内共生体衍生的信使 RNA (mRNA) 可以被宿主 RNAi 系统加工成 23-nt sRNA。我们预测了内共生体和宿主 mRNA 之间共享序列同一性的多个区域，并通过递送合成的内共生体 sRNA 证明，暴露于这些区域可以抑制互补宿主基因的表达，从而导致宿主生长成本。这种宿主基因敲低响应宿主 RNAi 因子对内共生体衍生的 RNA 加工的过程，我们称之为“RNAi 碰撞”，代表了一种可以促进兼性真核生物 - 真核生物内共生稳定性的机制。具体来说，