Identification of miRNA targets in hop (Humulus lupulus L.) in response to Verticillium nonalfalfae infection using degradome sequencing

Verticillium wilt is a vascular plant disease caused by soil-borne fungi of the genus Verticillium sensu stricto. Verticillium species infect many agriculturally important plants, including hops ( Humulus lupulus L.), which are cultivated for the brewing and pharmaceutical industries. In our previous study, we investigated the interaction between hops and species Verticillium nonalfalfae at the miRNA level and identified miRNAs showing differential expression between resistant and susceptible hop cultivars during the early phase of infection. In the present study, we performed hop degradome sequencing using two libraries per cultivar, prepared separately from mock-inoculated and V. nonalfalfae -inoculated plants, each library consisting of pooled samples collected at multiple time points during infection. In total, 399 target genes for 76 miRNAs were identified, of which 142 were detected exclusively in the resistant cultivar. Gene Ontology and KEGG analyses revealed that resistant-specific targeted genes are mainly involved in transcriptional regulation, protein metabolism, and stress responses, with enrichment in plant–pathogen interaction pathways. Of the seven degradome-identified miRNA-targeted genes analyzed, expression profiling revealed cultivar-dependent temporal responses. LOV1 was significantly downregulated in both cultivars, with an earlier response in susceptible Celeia (3 dpi) than in resistant Wye-Target (6 dpi), whereas THE1 displayed significant temporal changes only in Celeia. Additional target genes displayed temporal expression trends, although these were generally not statistically significant. These results provide valuable insights into miRNA-mediated regulatory mechanisms underlying hop defense responses to V. nonalfalfae infection.