RNA‐dependent RNA polymerase 1 delays the accumulation of viroids in infected plants

Abstract RNA‐dependent RNA polymerase 1 (RDR1) is essential for plant antiviral defence, but its role in plant defence against viroid infection remains unknown. The present study aimed to identify the function and mechanism of RDR1 in plant resistance to viroid infection. Overexpression of Nicotiana tabacum RDR1 (NtRDR1) delayed the accumulation of potato spindle tuber viroid (PSTVd) genomic RNA and PSTVd‐derived small RNA (sRNA) in Nicotiana benthamiana plants at the early invasion stage, but not in the late stage of infection. Conversely, virus‐induced gene silencing of tomato RDR1 (SlRDR1a) increased the susceptibility to PSTVd infection (increased viroid accumulation). Salicylic acid (SA) pretreatment induced SlRDR1a expression and enhanced the defence against PSTVd infection in tomato plants. Our study demonstrated that RDR1 is involved in SA‐mediated defence and restricts the early systemic invasion by PSTVd in plants. The decreased PSTVd accumulation in N. benthamiana was not caused by efficient accumulation of PSTVd sRNAs. These results deepen our understanding of the mechanism of RDR1 in plant defence responses to viroid attack.

2018; Qin et al., 2017;Xie et al., 2001;Yu et al., 2003). In addition, the expression of RDR1 is induced by plant hormones such as salicylic acid (SA) (Xie et al., 2001;Yu et al., 2003) and can be modulated by plant microRNAs . Previous studies found that plants were more sensitive to virus infection when RDR1 expression was repressed (Yu et al., 2003). In Nicotiana tabacum NtRDR1 gene knockout mutants, inoculation with tobacco mosaic virus (TMV) resulted in higher accumulation of TMV genomic RNA and more severe symptoms compared with those in wildtype plants (Xie et al., 2001).
Silencing of NtRDR1 transcription using a double-stranded, NtRDR1derived RNA hairpin resulted in increased accumulation of potato virus Y (PVY) RNA after infection (Rakhshandehroo et al., 2009).
Notably, the Nicotiana benthamiana NbRDR1 gene has a 72 bp insertion that prematurely terminates the gene's translation. This disruption of translation implies a natural loss-of function of the RDR1 in N. benthamiana that has been suggested to be the cause of the extreme susceptibility of N. benthamiana to a wide range of viruses, and makes these plants an excellent tool for plant molecular virologists (Bally et al., 2018;Yang et al., 2004). This phenomenon also verifies the antiviral function of RDR1 from another aspect. N. benthamiana plants transformed with RDR1 genes from Medicago truncatula and pepper (Capsicum annuum) are more resistant to infection by TMV, turnip vein-clearing virus, and sunn-hemp mosaic virus (Qin et al., 2017;Yang et al., 2004). In addition, RDR1 induction is involved in regulating symptom recovery after virus infection in tobacco (Basu et al., 2018). The antiviral properties of RDR1 have also been found in A. thaliana and cucumber. The AtRDR1 knockout mutant accumulates higher and more persistent levels of viral RNAs in infected leaves than those in wildtype plants (Yu et al., 2003). Multiple RDR1 genes are involved in virus resistance in cucumber and are regulated in a coordinated fashion with different expression profiles, for example constitutive expression of RDR1 in a transgenic cucumber line (Cucumis sativus) causes broad resistance to potyviruses (Leibman et al., 2011). These studies confirmed that RDR1 plays an important role in plant antiviral defence. However, in some cases RDR1 showed no significant correlation with plant antiviral defence. Silencing of the potato (Solanum tuberosum) RDR1 gene (StRDR1) did not increase potato susceptibility when challenged with three viruses: PVY, potato virus X (PVX), and TMV (Hunter et al., 2016). Surprisingly, N. benthamiana transformed with RDR1 from N. tabacum (NtRDR1) exhibit hypersusceptibility to plum pox virus and other viruses (Ying et al., 2010). This highlights the complexity of plant antiviral defence mechanisms. Plants must have different antiviral regulation genes and pathways that might interact and cooperate with each other.
For example, reduced accumulation of NtRDR1 in N. tabacum results in lower expression of other antiviral defence-related genes after PVY infection, such as RNA-dependent RNA polymerase 6 (RDR6), which is involved in RNA silencing (Rakhshandehroo et al., 2009).
Consequently, to fully determine the antiviral function of RDR1, we need to explore its molecular mechanism. Arabidopsis mutants lacking RDR1 produce lower levels of virus-derived small interfering RNAs (siRNAs), making them more susceptible to turnip mosaic virus (TuMV) and CMV infection. This suggests that RDR1 enhances plant viral resistance by amplifying virus-derived siRNAs, thus enhancing plant RNA silencing (Garcia-Ruiz et al., 2010;Wang et al., 2010).
Virus infection triggers widespread silencing of Arabidopsis genes by producing abundant endogenous siRNAs in plants, and this broadspectrum host antiviral activity depends on RDR1 expression (Cao et al., 2014). RDR1 also regulates plant genes involved in hormone synthesis and DNA methylation, suggesting that RDR1 may regulate plant antiviral defences in multiple ways (Lam et al., 2012;Stroud et al., 2013;Wang et al., 2014). Thus, RDR1 not only regulates plant viral resistance, but also acts as an intersecting node of plant gene expression regulatory pathways, such as RNA silencing and signal transduction.
In comparison with plant viruses, we know little about RDR1's involvement in the interaction between viroids and their hosts. Only a few viroids are known to induce RDR1 expression in plants (Campos et al., 2014;Schiebel et al., 1998;Xia et al., 2017). Therefore, it is necessary to study the role and mechanism of RDR1 in plant antiviroid defence. Our previous study showed that infection with hop stunt viroid (HSVd) induces RDR1 expression in cucumber (Cucumis sativus) (Xia et al., 2017); however, whether RDR1 induction is a common phenomenon in plants responding to viroid infection is unclear.
Consequently, we aimed to use combinations of PSTVd and tomato/ tobacco to identify the function and mechanism of RDR1 in plant resistance to viroid infection. This study will improve our understanding of viroid-host interactions and provide new ideas and methods to prevent and control viroid diseases.
The results showed that the EC samples contained a single amplicon, whereas the NtRDR1 samples contained two amplicons that differed by approximately 72 nt ( Figure S1). Western blotting of myc-NtRDR1 in transgenic N. benthamiana showed high amounts of myc-NtRDR1 in the total protein extracted from NtRDR1 leaf tissue, but none in the EC tissues (Figure 1c, Figure 1b). Moreover, the results of northern blotting were similar to those of the RT-qPCR assays.
The intensity of PSTVd genomic RNA signals was lower in NtRDR1 transgenic plants than in EC plants at 14 dpi, but this intensity difference was almost indistinguishable at 28 dpi (late infection stage) (Figure 1c).  (Wassenegger & Krczal, 2006). In our study, PSTVd genomic RNA accumulation was suppressed in transgenic NtRDR1 N. benthamiana plants. Whether the suppressed accumulation of PSTVd genomic RNA correlates with the production of PSTVd sRNA is unknown; therefore we analysed the accumulation of PSTVd sRNA in PSTVd-infected EC and NtRDR1 transgenic lines at 14, 21, and 28 dpi. At 14 dpi, a marked intensity of the PSTVd sRNA signals was observed in the EC lines but not in the NtRDR1 lines. Increasing accumulation of PSTVd sRNA was observed in the NtRDR1 lines at 21 and 28 dpi, which was always lower than in the EC lines ( Figure 2a).  Table S2).
However, the differences in the sRNA hot-spot patterns on both PSTVd strands indicated the existence of some unknown factors determining these differential profiles.

| Downregulation of RDR1a in tomato increased susceptibility to PSTVd infection
Tomato can be infected with PSTVd, and infected tomatoes exhibit severe leaf curling with vein necrosis and plant dwarfing, seriously threatening tomato fruit production (Diermann et al., 2010). Among the identified RDRs in S. lycopersicon, SlRDR1a shared the highest amino acid sequence (86%) identity with NtRDR1 (Liao et al., 2014). Despite this high sequence homology between SlRDR1a and NtRDR1, little is known about the roles of SlRDR1a in the tomato response to PSTVd infection ( Figure S3). Therefore, we explored the function of SlRDR1a in tomato. First, tobacco rattle virus (TRV)induced gene silencing was used to suppress SlRDR1a expression in tomato. pTRV1, along with pTRV2:SlRDR1a, was transformed into

| RDR1 is involved in SA-mediated defence against PSTVd infection
The signalling molecule salicylic acid (SA) plays important roles in both compatible and incompatible plant interactions with pathogens, including plant viruses (Dempsey et al., 1999). SA plays an important role in plant antiviral defence and RDR1 is involved in the SA-induced defence response to virus infection (Liao et al., 2014;Yang et al., 2004). A previous study showed that citrus exocortis viroid ( (Flores et al., 2015(Flores et al., , 2017 (Papaefthimiou et al., 2001). The synergism of DCL2 and DCL3 in N. benthamiana strongly suppressed PSTVd accumulation (Katsarou et al., 2016).  might also regulate antiviral defence via hormones (Lam et al., 2012).
SA plays a critical role in plant defence against pathogen attack, mainly via two pathways: RDR1-mediated RNA silencing and the alternative oxidase (AOX)-associated defence pathway. The application of an exogenous AOX activator on tomato plants markedly induces the accumulation of SlRDR1 and SlAOX1a transcripts and reduces TMV RNA accumulation, indicating that RDR1 is involved in the AOXmediated defence pathway against TMV infection (Liao et al., 2014).
Furthermore, exogenous SA application on N. tabacum induces rapid nitric oxide (NO) accumulation, which functions upstream of H 2 O 2 to mediate RDR1 induction, playing a critical role in restricting virus systemic infection and accumulation (Liao et al., 2013). In the present study, SA pretreatment induced SlRDR1a expression rapidly, and this downstream response enhanced the defence against PSTVd infection in tomato plants ( Figure 6). In contrast, SA did not increase SlRDR1a transcript abundance in SlRDR1a-silenced plants and, interestingly, the resistance of SlRDR1a-silenced tomato to PSTVd was partially restored by exogenous SA application ( Figure 6). This suggests that SA might regulate multiple pathogen defence pathways in tomato, such as other silencing-related genes like DCL1, DCL2, RDR1, and RDR2 in tomato that are induced after SA treatment (Campos et al., 2014) or genes related to an AOX-associated defence pathway (Liao et al., 2014). Thus, further experiments to determine the other factors involved in SA-associated induction will reveal the mechanisms of plant antiviroid defence reactions.
In summary, we provided evidence of crosstalk between RDR1  (Ying et al., 2010).

| Plasmid construction and inoculation
PSTVd-s (GenBank accession no. MK303581) was used in our experiments. pGEM-PSTVd plasmid contained head-to-tail tandem PSTVd cDNA repeats. The linearized fragment of pGEM-PSTVd was digested using SpeI (Takara), and then the PSTVd dimer RNA plus-strand was transcribed using T7 RNA polymerase (Promega). BamHI sites to generate pCAM1300-CsRDR1c1-GFP, which was transformed into A. tumefaciens GV3101 and used for genetic transformation. The primers used are shown in Table S1.
The VIGS constructs were generated following the method of Liao et al. (2014). A fragment of the coding region of SlRDR1a (approximately 400 nt) was cloned into vector pTRV2 between the  Liu et al. (2002).
After viral infection, the plants were maintained in a greenhouse (26 ℃) before use. PCR fragments were amplified using the primers shown in Table S1. All constructs were confirmed by sequencing

| Northern blotting and siRNA-blot hybridization
Total sRNAs were extracted using an miRcute miRNA Isolation Kit (Tiangen). Northern blotting was performed as previously described . In brief, DIG-labelled cRNA probes for PSTVd RNA were obtained by in vitro transcription using a DIG RNA labelling kit (Roche Applied Science) according to the manufacturer's instructions.
Equal loading was confirmed by 5S rRNA fluorescence after ethidium bromide staining and UV irradiation (Tanon 2500).

| Protein extraction and western blotting hybridization
Protein extraction and western blot hybridization were performed according to Li et al. (2018). After transferring protein to nitrocellulose membranes, the membranes were probed with a mouse anti-GFP or anti-myc antibody and then probed with a horseradish peroxidase-labelled goat anti-mouse antibody. Detection signal was visualized using the EasySee Western Blot Kit (TransGen Biotech) according to the manufacturer's protocol.

| Deep sequencing and sequence analysis of viroid derived-sRNAs
Uninoculated systemic leaves collected at 21 days after PSTVd inoculation were collected for sRNA sequencing. sRNA samples were prepared use TruSeq Small RNA Sample Prep Kits (Illumina) and sequenced using an Illumina Hiseq 2000/2500 instrument; the sequencing read length was 1 × 50 bp. ACGT101-miR (LC Sciences) was used to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA), and repeats from the raw reads. The project was carried out by Lc-Bio Technologies (Hangzhou) Co., Ltd. The obtained sRNA sequences (21-24 nt) were mapped to the PSTVd genome, and the circularity of the viroid genome was taken into consideration. For further analysis, the 21-24 nt sequences were pooled, and each set of sequences was analysed by BLAST searching against the nucleotide sequence of the PSTVd-s strain. No mismatch was allowed. Data were analysed and visualized for specific distribution patterns and phasing (WPS Office Excel 2019).

| Data analysis
All data were analysed using Student's t test (n = 6), with three independent replicates in each experiment. In the figures, ** indicates statistically significant differences compared with the control at p < 0.01 and different letters indicate significant differences between treatments (p < 0.05).

ACK N OWLED G EM ENTS
The authors are grateful to Professors Teruo Sano and Nuredin Habili for their help with the writing and valuable comments.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.