Genome‐wide analysis of small RNAs from Odontoglossum ringspot virus and Cymbidium mosaic virus synergistically infecting Phalaenopsis

Summary Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the two most prevalent viruses infecting orchids and causing economic losses worldwide. Mixed infection of CymMV and ORSV could induce intensified symptoms as early at 10 days post‐inoculation in inoculated Phalaenopsis amabilis, where CymMV pathogenesis was unilaterally enhanced by ORSV. To reveal the antiviral RNA silencing activity in orchids, we characterized the viral small‐interfering RNAs (vsiRNAs) from CymMV and ORSV singly or synergistically infecting P. amabilis. We also temporally classified the inoculated leaf‐tip tissues and noninoculated adjacent tissues as late and early stages of infection, respectively. Regardless of early or late stage with single or double infection, CymMV and ORSV vsiRNAs were predominant in 21‐ and 22‐nt sizes, with excess positive polarity and under‐represented 5ʹ‐guanine. While CymMV vsiRNAs mainly derived from RNA‐dependent RNA polymerase‐coding regions, ORSV vsiRNAs encompassed the coat protein gene and 3ʹ‐untranslated region, with a specific hotspot residing in the 3ʹ‐terminal pseudoknot. With double infection, CymMV vsiRNAs increased more than 5‐fold in number with increasing virus titres. Most vsiRNA features remained unchanged with double inoculation, but additional ORSV vsiRNA hotspot peaks were prominent. The potential vsiRNA‐mediated regulation of the novel targets in double‐infected tissues thereby provides a different view of CymMV and ORSV synergism. Hence, temporally profiled vsiRNAs from taxonomically distinct CymMV and ORSV illustrate active antiviral RNA silencing in their natural host, Phalaenopsis, during both early and late stages of infection. Our findings provide insights into offence–defence interactions among CymMV, ORSV and orchids.