Comparative small RNA profiles of beet mosaic virus (BtMV), beet mild yellowing virus (BMYV) and beet yellows virus (BYV) infected Nicotiana benthamiana and Beta vulgaris

Plants are constantly challenged by viral pathogens that can limit growth and reduce yield. A key component of the plant innate immunity is RNA silencing, in which viral double-stranded RNA (dsRNA) intermediates are recognised and processed into virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs direct the degradation of viral genomes, thereby restricting infection. Sugar beet (Beta vulgaris subsp. vulgaris) is a crop of major economic importance, where the virus yellows (VY) complex represents a serious threat to production. Here, we profiled and compared vsiRNAs generated during infection of the natural host plant B. vulgaris and the experimental host plant Nicotiana benthamiana with three taxonomically distinct viruses: beet yellows virus (BYV, Closterovirus), beet mild yellowing virus (BMYV, Polerovirus), and beet mosaic virus (BtMV, Potyvirus). High-throughput sequencing of small RNAs revealed characteristic size distributions and strand biases that differed among viruses and host species. Comparative analysis highlighted no host plant-specific pattern of vsiRNA accumulation. This comparative approach provides a detailed view of vsiRNA processing and offers novel insights that are not apparent from coverage profiles alone. Distinct vsiRNA hotspots were detected for each viral genome, and these hotspots did not differ between host plants, pinpointing potential target regions for RNA interference-based control approaches. The identification of such regions provides a basis for the design of synthetic dsRNAs that can be applied exogenously as protective sprays, an emerging, non-transgenic strategy to mitigate VY infections, while advancing understanding of vsiRNA biogenesis in sugar beet and N. benthamiana in general.