In plants, RNA interference (RNAi) generates small interfering (si)RNAs from entire genomes of viruses, satellites and viroids. Therefore, deep small (s)RNA sequencing is a universal approach for virome reconstruction and RNAi characterization. We tested this approach on dried barley leaves from field surveys. Illumina sequencing of sRNAs from 2 plant samples identified in both plants Hordeum vulgare endornavirus (HvEV) and barley yellow mosaic bymovirus (BaYMV) and, additionally in one plant, a novel strain of Japanese soil-borne wheat mosaic furovirus (JSBWMV). De novo and reference-based sRNA assembly yielded complete or near-complete genomic RNAs of these viruses. While plant sRNAs showed broad size distribution, viral sRNAs were predominantly 21 and 22 nucleotides long with 5'-terminal uridine or adenine, and were derived from both genomic strands. These bona fide siRNAs are presumably processed from double-stranded RNA precursors by Dicer-like (DCL) 4 and DCL2, respectively, and associated with Argonaute 1 and 2 proteins. For BaYMV (but not HvEV, or JSBWMV), 24-nucleotide sRNAs represented the third most abundant class, suggesting DCL3 contribution to antibymovirus defence. Thus, viral siRNAs are well preserved in dried leaf tissues and not contaminated by non-RNAi degradation products, enabling both complete virome reconstruction and inference of RNAi components mediating antiviral defense.Read less <