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

Identification of CymMV and ORSV infection in P. equestris. P. equestris samples showing severe chlorotic, ringspots even necrotic symptoms (Fig. 1A) caused by CymMV and ORSV infection about 15 days were collected from Orchid Cultivation Distribution Center in Zhangzhou, Fujian. The co-infection of CymMV and ORSV was confirmed by RT-PCR and by H-7650 Hitachi transmission electron microscopy. Our results showed that coat protein genes of CymMV and ORSV were successfully amplified by RT-PCR (Fig. 1B) . Furthermore, both negative staining and thin sections suggested that abundant of approximately 300-nm rod-shaped and 500-nm linear virus particles, representing for CymMV and ORSV, respectively, existed in viruses-infected P. equestris plants ( Fig. 1C and D) . Characterizations of siRNAs derived from CymMV and ORSV in P. equestris. Following confirmation of CymMV and ORSV co-infection, sRNA from virus-infected and virus-free plant samples was sequenced with the Solexa protocol. Totally, 6,133,689 and 7,563,892 clean reads were obtained from the sRNA libraries of virus-infected and virus-free sample, respectively. Among these reads, a total of 369,524 (6.02%) and 457,104 (7.45%) reads from virus-infected sample were mapped to the CymMV and ORSV genomes, respectively. Conversely, only 1,007 (0.01%) reads matched the two virus genomes in virus-free sample. 21-nt reads increased, while 24-nt reads decreased significantly in the virus-infected sample compared with virus-free sample ( Fig. 2A ), suggesting that virus infection changed the distribution pattern of sRNA. In virus-infected sample, the majority (72% and 66%) of siRNAs derived from CymMV and ORSV were 18-22 nt in length, with 21 and 22-nt being most abundant ( Fig. 2B and C); however, the majority of siRNAs in many virus-plant systems were 21-24 nt in length, with 21-and 22-nt being also most abundant (Kreuze et al., 2009; Lan et al., 2018b; Li et al., 2016; Mitter et al., 2013; Xu and Zhou, 2017; Yan et al., 2010; Yang et al., 2014) . Thus, these results implicated that the homologues of DCL4 and DCL2 in P. equestris may be the predominant DCLs involved in the biogenesis of viral small interfering RNAs (vsiRNAs) which functions as the predominant antiviral silencing mediators (Blevins et al., 2006; Deleris et al., 2006; Ding, 2010; Donaire et al., 2008; Liu et al., 2018; Niu et al., 2017) ; as for other DCLs of P. equestris participating in vsiRNAs generation will be the future discussed topic. Then, we analyzed the features of 21-nt siRNA duplexes induced by CymMV and ORSV. The results showed that 21-nt siRNA duplexes derived from CymMV and ORSV with 0-nt overhangs were the most abundant 21-nt duplexes, followed by 2-nt overhangs and then 1-nt overhangs 21-nt duplexes in P. equestris (Fig. 2D) , which was different from that of other virusesplant and viruses-invertebrates systems, in which the 21nt siRNAs duplexes with 2-nt overhands were the most abundance duplexes (Liu et al., 2018; Niu et al., 2017) . Thus, the results suggested that 21-nt vsiRNAs duplexes with 0-nt overhangs were the most efficient triggers of RNAi in P. equestris. It has been reported that sRNA sequences could be overlapped and further assembled into longer transcript contigs (Kreuze et al., 2009 ). Our results revealed that 4 and 7 transcript contigs, with a longest contigs of 3,167 and 2,853 nt, were obtained respectively for CymMV and ORSV with the short sequence assembly program Velvet ( Fig. 3A and B) . Th