以高通量定序方式分析蝴蝶蘭感染齒舌蘭輪斑病毒及蕙蘭嵌紋病毒後小型核醣核酸組成之變化

Abstract

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most prevalent viruses infecting orchids and causing economic losses worldwide. Upon virus infection, small RNA mediated antiviral RNA silencing response is activated. Such biotic stress may affect virus-specific interfering RNAs (vsRNAs) or microRNA (miRNA) regulated host gene expression. To advance our understanding of the offense-defense interactions between CymMV, ORSV and orchids, this study employed deep sequencing to analyze small RNAs from virus infected Phalaenopsis. The leaf tip-inoculation method first distinguished early and late stages of infection in non-inoculated and inoculated tissues at ten days post inoculation (dpi). Small RNA Solexa sequencing generated 11 libraries with more than five million reads each from CymMV and ORSV singly and doubly inoculated leaves and from mock-inoculated leaves. Generally, CymMV and ORSV vsRNAs were predominantly 21 and 22 nucleotides (nt), with excess positive polarity accumulating in single inoculations. While most CymMV vsRNAs were derived from RNA-dependent RNA polymerase (RdRp) coding regions, ORSV vsRNAs encompassed the coat protein coding gene and 3’-untranslated region, with a specific hotspot residing in the pseudoknot upstream to the 3’-terminal tRNA-like structure. These results suggest Dicer-like (DCL) 4 and DCL2 homologs play a leading role in mediating antivirus RNA silencing in P. amabilis using single-stranded RNA derived secondary structure as templates. Biased distribution of 5’ terminal adenosine (A), uridine (U), cytosine (C) and underrepresented 5’-guanine (G) indicate vsRNAs could be recruited into multiple Argonaute (AGO) complexes. Under mixed infection, chlorotic necrosis symptoms appeared specifically in inoculated tissues at 10 dpi, and accelerated spreading and enhanced viral titer of CymMV also occurred. The proportion of CymMV vsRNAs in total small RNAs ranged from 5.83% in singly infected tissues to 27.9% in doubly infected tissues, providing evidence of the enhancement of CymMV titer. While most vsRNA features remained unchanged in double inoculations, three additional prominent ORSV vsRNA hotspot peaks were observed. In silico prediction revealed Phalaenopsis transcript hotspots that are potential targets for vsRNA are also likely to be involved in symptom formation. The virus infection also modulated miRNA expression—for example, miR156, miR168, miR894 were up regulated and miR398, miR408, miR528 were down regulated after CymMV or ORSV infection. These infection responsive miRNAs participate in a broad spectrum of cellular processes like hormone and metabolite assimilation, signal transduction, and oxidative stress calibration. Taken together, the deep sequencing provided a global profile of vsRNAs and miRNAs in Phalaenopsis under CymMV and ORSV infection. Further research should provide valuable insights into small RNA-mediated virus-plant interactions.