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

To further confirm the response of RNAi pathway to CymMV and ORSV infection, we obtained the sequences of the RNAi pathway core components in the genome of P. equestris through a local BLASTz search with corresponding sequences of O. sativa as queries (Cai et al., 2015; Kapoor et al., 2008; Niu et al., 2016) . We then investigated the transcript levels of these core components by RT-qPCR assay on a QuantStudio 6 Flex Real-Time PCR System (Life Technologies) using the SYBR Green PCR MasterMix kit (Promega) as described previously (Lan et al., 2015 (Lan et al., , 2018a . In total, we identified 14 core components of RNAi pathway in P. equestris genome, including DCL1, DCL2a, DCL3a, DCL4, AGO4B, AGO7, AGO10, AGO16, AGO-PNH1, AGO-MEL1, RdRP1, RdRP2, RdRP5, and RdRP-SHL2 ( Supplementary Fig. 2 ). As indicated in Fig. 4 , RT-qPCR assays demonstrated that DCL2a, DCL4, AGO4B, AGO7, AGO10, RdRP1, and RdRP5 transcript levels increased significantly by 2-6 folds (Fig. 4) ; in contrast, transcript levels of DCL1, AGO16, and RdRP-SHL2 decreased obviously by 50-70% (Fig. 4) in virus-infected P. equestris compared with virus-free P. equestris. Transcript levels of other genes of RNAi pathway change slightly (Fig. 4) . Researches have shown that down-regulation of DCL2 and DCL4 in virus-infected rice may influence the production of 21-and 22-nt vsiRNAs and thus weaken host defense, allowing viruses to establish successful infection. For example, mutation of DCL4 from O. sativa can induce abnormal expression of auxin response factors and lead to growth abnormities (Liu et al., 2007) . In our study, however, DCL2a and DCL4 were up-regulated but DCL1 was down-regulated (Fig. 4) . Thus, the down-regulation of DCL1 in virus-infected P. equestris might partially account for P. equestris symptoms showing severe chlorotic, ringspots even necrotic. DCL2a and DCL4 may have additional biological pathways or functions. Many viral suppressors of RNA silencing (VSRs) can block biogenesis of vsiRNAs by interacting with plant DCLs. For example, DCL4 from O. sativa is suppressed by RYMV P1 (Lacombe et al., 2010) . It is yet to confirm whether VSR proteins encoded by CymMV and ORSV interacts with the DCL1 of Fig. 4 . Core components of RNA interference (RNAi) were selectively and differently modulated by infection of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) in Phalaenopsis equestris. Relative transcript levels of core components of RNAi in CymMV and ORSV co-infected and viruses-free P. equestris as detected by real-time reverse transcription polymerase chain reaction assay with mean ± standard deviation (SD) of three independent experiments. Statistical analysis was conducted based on Tukey's honest significant difference test using SAS version 4 (SAS Institute, Cary, NC, USA). **P < 0.01, *P < 0.05. DCL, Dicer-like; RdRP, RNA-dependent RNA polymerase. P. equestris. Plant AGOs participate in host defense against many viruses species. In our study, CymMV and ORSV infection-induced up-regulation of AGO4B, AGO7, and AGO10 but down-regulation of AGO16 (which was predicted as a target of a certain vsiRNA, as after-mentioned) (Fig. 4) , and in particular, AGO4B and AGO7 was drastically up-regulated (Fig. 4) , suggesting facilitated host antiviral defense. The dramatically activated AGO4B and AGO7 might not only act in vsiRNAs binding, but also have additional biological functions. Whether the downregulated AGO16 in virus-infected plant affected R