Cucumber extracellular vesicles-localized tetraspanin and phloem protein 2 mediates the cross-kingdom trafficking of a pathogenic non-coding RNA

The plant apoplast and Extracellular Vesicles (EVs) harbor various RNA types, including small RNAs, long non-coding RNAs, and circular RNAs that traverse kingdom boundaries via encapsulation within EVs. Viroid RNAs, which are pathogenic circular non-coding RNAs, move across kingdoms, from plants to insects and fungi. However, the molecular mechanism underlying this cross-kingdom trafficking remains largely elusive. Using apple scar skin viroid (ASSVd) as a model for circular non-coding RNAs and cucumber as its host, we show that viroid RNAs, in their circular, linear, and viroid-derived small RNA forms, are present in cucumber EVs. Additionally, we revealed that ASSVd RNA exhibits extracellular RNA signatures, such as m6A methylation and five EXOmotifs, aiding their packaging into EVs. Our characterization of the cucumber EV proteome validated the role of tetraspanin 8 (TET8) as a conserved marker protein in cucumber EVs. Additionally, we report PP2-a phloem lectin with RNA binding domain as a novel protein associated with cucumber EVs. Through RNA-Co-immunoprecipitation and immunoenrichment assays, we show that PP2 and ASSVd are present in TET8-specific exosomes, with direct interactions occurring between PP2-TET8 and PP2-ASSVd. We propose that PP2 may act as an RNA sorting protein, aiding in the RNA loading into EVs. The potential for cross-kingdom viroid RNA transmission via EVs was also investigated and confirmed. In conclusion, our findings suggest that viroid RNAs could serve as a model RNA system for exploring the complexities of cross-kingdom RNA trafficking in plants.