Selective packaging of viroid RNAs into plant exosomes, mediated by host proteins, enables efficient cross-kingdom transmission

Background: The plant apoplast and extracellular vesicles (EVs) contain diverse RNA species, including small RNAs, long non-coding RNAs, and circular RNAs, many of which cross kingdom boundaries through EV-mediated transport. Viroid RNAs particularly Apple Scar Skin Viroid (ASSVd) are known to move from plants to fungi and insects, though the mechanism remains unclear. Results: Using cucumber as a host and Apple scar skin viroid (ASSVd) as the pathogen, we demonstrate through RT-PCR and northern blot analyses that multiple forms of viroid RNAs are present within cucumber-derived EVs. Transmission assays show that these EVs retain viroid infectivity, successfully transmitting the viroid to healthy plants and whiteflies via artificial diets supplemented with viroid-positive EVs. Proteomic analyses identify tetraspanin 8 (CsTET8) as a conserved EV marker, and reveal CsPP2—a phloem lectin with RNA-binding capacity—as a novel EV-associated protein. Co-immunoprecipitation and enrichment assays further confirm the co-localization of CsPP2 and ASSVd within CsTET8-positive EVs, along with direct interactions between CsPP2, CsTET8, and ASSVd. Conclusions: In summary, this study demonstrates that cucumber-derived EVs can carry infectious ASSVd RNA, which remains transmissible to healthy plants and insect vectors under experimental conditions. The EVs are marked by CsTET8, and CsPP2 is identified as a novel RNA-binding protein associated with viroid-containing EVs, directly interacting with both CsTET8 and ASSVd. These findings propose a potential EV-mediated pathway for viroid movement and transmission in plants and establish viroid RNAs as a model system to study the molecular basis of RNA trafficking across biological kingdoms.