siRNAs Derived from Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus Down-modulated the Expression Levels of Endogenous Genes in Phalaenopsis equestris

Lan, Han-hong; Wang, Cui-mei; Chen, Shuang-shuang; Zheng, Jian-ying

Previous studies have reported that the 5′-terminal nucleotide of sRNAs play irreplaceable roles in mediating the sorting of sRNAs into AGO complexes in plants (Mi et al., 2008) . Consistent with previous report for diverse plant-virus-specific sRNAs (Donaire et al., 2008; Lan et al., 2018b; Xu and Zhou, 2017) , CymMV and ORSV siRNAs also demonstrated a clear tendency to begin with uracil (U), adenine (A), as compared with cytosine (C) and guanidine (G) (Fig. 2E and F) . To explore the origin of vsiRNAs, their polarity distribution was further characterized. Our results showed that CymMV and ORSV siRNAs were derived predominantly from the viral positive-strand RNA, accounting for about 90% (Fig. 2G and H) . This is broadly contrasted to many single-stranded RNA (ssRNA) positivestrand viruses, siRNAs of which were produced equally from the positive and the negative strands or predominantly from negative strands of viral genomes (Ho et al., 2007; Lan et al., 2018b; Li et al., 2016; Xia et al., 2014; Yang et al., 2014) . Furthermore, single-base resolution maps of total CymMV and ORSV siRNAs along with genomes were created using Bowtie tools and in-house Perl scripts. Our results revealed that majority siRNAs were from the intergenic regions, not the 5′-or 3′-terminal regions of the two viruses genomes and siRNAs had a continuous but heterogeneous (Hot spot and Cold spot) distribution along the genomes of the two viruses ( Fig. 3A and B) . Taken together, our results implied that highly structured regions present in CymMV and ORSV genomes may be the substrates for DCL enzymes. To explore the possibility, we evaluated the secondary structures of CymMV and ORSV with RNAfold server. Our results showed that lots of stem-loop secondary structures were formed in intergenic regions and corresponded to the hotspots ( Fig. 3C and D) . Taken together, these results supports that siRNAs should originate predominantly by direct DCL cleavage of imperfect duplexes in the most folded regions of the positive strand of both viruses RNA molecular in P. equestris, similar to the speculation for siRNAs origin from plant infected with Cymbidium ringspot tombusvirus (CymRSV), a ssRNA positive-strand virus, implying siRNAs in the CymRSV system are also predominantly processed by DCL cleavage of imperfect duplexes, which can be formed from the positive-strand viral RNA (Molnár et al., 2005) .