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) 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--pathogenic circular non-coding RNAs--are known to move from plants to fungi and insects, though the mechanism remains unclear. Using apple scar skin viroid (ASSVd) and cucumber as a host, we demonstrate that viroid RNAs in circular, linear, and small RNA forms are present in cucumber EVs. ASSVd RNA exhibits features of extracellular RNAs, such as m6A methylation and five EXOmotifs. Through mass spectrometry and immunoblotting, we identify tetraspanin 8 (CsTET8) as a conserved EV marker and report CsPP2, a phloem lectin with an RNA-binding domain, as a novel EV-associated protein. RNA co-immunoprecipitation and immuno-enrichment assays reveal that CsPP2 and ASSVd co-localize in CsTET8-positive EVs, with direct interactions between CsPP2-CsTET8 and CsPP2-ASSVd. We propose CsPP2 functions as an RNA-sorting protein that mediates RNA loading into EVs. Further, we show that whiteflies can acquire ASSVd via EVs and transmit it to healthy plants, indicating a cross-kingdom transmission route. These findings establish viroid RNAs as a model system to study the molecular basis of RNA trafficking across biological kingdoms.