Use of deep sequencing to profile small RNAs derived from tomato spotted wilt orthotospovirus and hippeastrum chlorotic ringspot orthotospovirus in infected Capsicum annuum.

Virus-derived small RNAs are one of the key factors of RNA silencing in plant defence against viruses. We obtained virus-derived small interfering RNA profiles from Tomato spotted wilt orthotospovirus and Hippeastrum chlorotic ringspot orthotospovirus infected Capsicum annuum XX19 and XY11 by deep sequencing one day after inoculation. The vsiRNAs data were mapped to the TSWV and HCRV genomes, and the results showed that the vsiRNAs measured 19-24 nucleotides in length. Most of the vsiRNAs were mapped to the S segment of the viral genome. For XX19 and XY11 infected with HCRV, the distribution range of vsiRNAs in S RNA was 52.06-55.20%, while for XX19 and XY11 infected with TSWV, the distribution range of vsiRNAs in S RNA was 87.76-89.07%. The first base at the 5' end of the siRNA from TSWV and HCRV was primarily biased towards A, U, or C. Compared with mock-inoculated XX19 and XY11, the expression level of CaRDR1 was upregulated in TSWV- and HCRV-inoculated XX19 and XY11. CaAGO2 and CaAGO5 were upregulated in XY11 against HCRV infection, and CaRDR2 was downregulated in TSWV-infected XY11 and XX19. The profile of HCRV and TSWV vsiRNA verified in this study could be useful for selecting key vsiRNA such as those in disease-resistant varieties by artificially synthesizing amiRNA.

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

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 5'-guanine. While CymMV vsiRNAs mainly derived from RNA-dependent RNA polymerase-coding regions, ORSV vsiRNAs encompassed the coat protein gene and 3'-untranslated region, with a specific hotspot residing in the 3'-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.