MicroRNAs in the exosome-like nanoparticles from orange juice inhibit Citrus blue mold caused by Penicillium italicum

Whether the exosomes in the juice can protect citrus fruit against pathogenic infection is largely unknown. In this study, extracellular vesicles were isolated and characterized from C. reticulata juice, and tested whether they could interact with spores of Penicillium italicum. Isolated EVs were characterized by nanoparticle tracking analysis and transmission electron microscopy. Membrane-labeled exosome-like nanoparticles (EXO-CRs) were prepared and their uptake by the spores of P. italicum was verified. It was found that the EXO-CRs could significantly inhibit the mycelium growth of P. italicum on citrus fruit. To explore the potential mechanisms underlying small RNA-mediated EXO-CR inhibition of the invading pathogens, we performed transcriptome, sRNA, and degradome sequencing to identify potential small RNA molecules that may contribute to such inhibitory effect of EXO-CRs. A total of 100 miRNAs from EXO-CRs were identified and target characterization was further unraveled by the Tobacco transient co-expression system. The results revealed that certain miRNA-target modules were closely associated with host defense-related pathways, including TCA cycle, spliceosome and ribosome function, and endocytosis. Taken together, we elucidated the molecular mechanism of citrus exosome inhibition on P. italicum by identifying the key miRNAs and their targets with critical roles in host plant-derived cross-kingdom pathogen RNA interference.