Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosan a

Abstract The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus‐induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids.

Many AGOs have been reported to mediate defence against viruses. For example, in Arabidopsis thaliana, AGO1 participates in the defence mechanism for restricting turnip crinkle virus (TCV) and cucumber mosaic virus (CMV) infections (Qu et al., 2008;Wang et al., 2011). In Nicotiana benthamiana, NbAGO1 was recently found to inhibit bamboo mosaic virus (BaMV) accumulation, and NbAOG10 may compete with NbAGO1 for BaMV-derived small interfering RNAs (vsiRNAs) to protect BaMV from NbAGO1-mediated antiviral RNA cleavage (Huang et al., 2019). AGO2 is involved in the defence against potato virus X (PVX), TCV, CMV, and turnip mosaic virus (TuMV) in various plants (Harvey et al., 2011). The abscisic acid-mediated up-regulation of AGO2 and AGO3 induces resistance to BaMV (Alazem et al., 2017). The deficiency of AtAGO4 predisposes A. thaliana plants to tobacco rattle virus (TRV) infection . AtAGO7 and AtAGO10 provide resistance against TCV and TuMV, respectively (Garcia-Ruiz et al., 2015;Qu et al., 2008). AGO18 is a grass-specific AGO subfamily and is close to the AGO1/5/10 clade. OsAGO18 from Oryza sativa was reported to be induced in rice infected by rice dwarf virus (RDV) and rice stripe virus (RSV), and is involved in the maintenance of OsAGO1 expression through the sequestration of miR168, which in turn improves the OsAGO1-mediated antiviral defence against RDV and RSV (Wu et al., 2015). Despite the wealth of studies on the importance of AGOs in antiviral mechanisms in the model plants mentioned above, the identities and functions of AGOs against viral infections in orchids have not been systematically analysed previously.
In this study, Phalaenopsis aphrodite subsp. formosana, the moth orchid native to Taiwan, was chosen as the representative orchid, based on economic importance and the availability of the genomic information (Chao et al., 2017), for the comprehensive analysis of the functions of AGOs against viral infections. Our previous study revealed that the accumulation levels of four AGOs (PaAGO1, 4, 5, and 10) in leaves varied significantly depending on the invading viruses (Pai et al., 2020). Here, we identified and analysed the expression profiles of all candidate PaAGOs in response to different viral infections. Subsequently, the PaAGO5 family proteins were targeted for further analysis on their roles in antiviral mechanisms by gene knockdown or overexpression because PaAGO5s were the only ones found to be responsive to viral infections in this study.
It was revealed that both PaAGO5a and PaAGO5b could enhance resistance against CymMV and ORSV. To our knowledge, this is the first comprehensive study of the AGOs in response to virus infections in P. aphrodite subsp. formosana, demonstrating the unique involvement of PaAGO5 family proteins against prevalent orchid viruses.

| Identification of the AGOs in P. aphrodite subsp. formosana
Although the transcriptomic information of P. aphrodite subsp. formosana is available (Chao et al., 2017), the genes encoding AGOs (PaAGOs) have not been thoroughly identified previously. To provide a comprehensive understanding of the AGOs involved in the defence responses against viruses, we performed exhaustive searches in different genomic databases using the sequences of known AGO genes from N. benthamiana and Arabidopsis thaliana as queries with the BLAST search (Altschul et al., 1990). The databases searched included GenBank (www.ncbi.nlm.nih.gov/genbank, Sayers et al., 2020) of the National Center for Biotechnology Information (NCBI) and Orchidstra 2.0 (Chao et al., 2017). In addition, keyword searches of annotated AGOs were also performed in these databases. A total of 11 PaAGOs were identified from the Orchidstra 2.0 database for orchid transcriptomes. To illustrate the evolutionary relationship with the AGOs of known model plants, we conducted a multiple sequence alignment using ClustalW (Thompson et al., 1994) followed by phylogenetic analysis of the 11 PaAGOs and representatives from A. thaliana (AtAGOs) (Berardini et al., 2015), using the general time reversible (GTR; Tavaré, 1986) substitution model and the maximum-likelihood method for phylogenetic tree reconstruction in MEGA X (Adachi et al., 2000;Kumar et al., 2018). In accordance with previous reports (Huang et al., 2019;Rodríguez-Leal et al., 2016), the 11 PaAGOs were grouped into AGO1, AGO4, AGO5, AGO6, AGO7, and AGO10 with the previous classification of AtAGOs subfamilies (Figure 1). The designations of the transcripts were as follows: PATC157237 (PaAGO1a), PATC157597 (PaAGO1b), PATC129162 (PaAGO5a), PATC139886 (PaAGO5b), PATC151309 (PaAGO5c), PATC139605 (PaAGO4), PATC127159 (PaAGO6), PATC003663 (PaAGO7a), PATC084109 (PaAGO7b), PATC130660 (PaAGO10a), and PATC093469 (PaAGO10b). From the transcript expression data in the Orchidstra 2.0 database, PaAGO1a showed the highest expression level among all PaAGOs in leaves ( Figure S1a). PaAGO1b and PaAGO4a exhibited a similar expression level, followed by PaAGO5b, which is the most abundant PaAGO5.
The expression level of the rest of the PaAGOs in descending order was PaAGO10b, PaAGO6, PaAGO10a, PaAGO7a, and PaAGO7b. The tissue-specific expression profile indicated that most of the PaAGOs were primarily distributed in flower, root, stalk, or seed (Figure S1b), with lower expression levels in leaf and polonium.
F I G U R E 1 Phylogenetic relationship of the Argonaute (AGO) family proteins of Phalaenopsis aphrodite subsp. formosana. The amino acid sequences of AGO proteins identified in P. aphrodite subsp. formosana, with those from Arabidopsis thaliana as references, were aligned using the ClustalW software (Thompson et al., 1994) and subjected to phylogenetic analysis using the maximum-likelihood method in MEGA software (Kumar et al., 2018) with 1,000 bootstrapping replicates using default parameters. The bootstrap values are shown next to the branches. Major clades are boxed in different colours according to the classification reported by Rodriguez-Leal et al. (2016). Scale bar, 0.2 substitutions per site F I G U R E 2 Accumulation of viral coat proteins (CP) at different intervals following infections by CymMV and/or ORSV in Phalaenopsis aphrodite subsp. formosana leaves. P. aphrodite subsp. formosana leaves were infiltrated with Agrobacterium tumefaciens EHA105 harbouring infectious clones of CymMV and ORSV, pKCy1 and pKORy-15-2, respectively, using the AGROBEST method (Wu et al., 2014), either alone or mixed, at an OD 600 of 0.5. The leaves were collected at 5 (a), 10 (b), 15 (c), and 20 (d) days postinoculation (dpi) for analysis. The protein extracts from leaves at different dilutions were electrophoresed through a 12.5% acrylamide gel containing 1% sodium dodecyl sulphate (SDS-PAGE), followed by western blot (WB) analysis using specific antibodies against CymMV or ORSV as indicated. The samples were diluted differently, as indicated, to accommodate the differences of each antibody. The intensity of each band was quantified and plotted. Coomassie blue-stained RuBisCO protein (CBS-RuBisCO) was used as the loading control. Samples from P. aphrodite subsp. formosana leaves agroinfiltrated with empty vector pKn only (EV), pKCy1 alone (CymMV), pKORy-15-2 alone (ORSV), or both infectious clones (Mixed) were as indicated. Vir, the mixture of virions containing 50 ng CymMV and 5 ng ORSV

| PaAGOs expression profiles following CymMV and/or ORSV infection
To unveil the roles of the PaAGOs in the antiviral defence responses, the expression profiles of PaAGOs during viral infections were examined. P. aphrodite subsp. formosana leaves at the four-to five-leaf stages were inoculated with CymMV and/or ORSV infectious clones through agroinfiltration. The plants were kept at 28 °C with a 15-hr light period and leaf samples were collected at 5, 10, 15, and 20 days postinoculation (dpi). The accumulation levels of PaAGO transcripts and viruses were analysed by quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR) and western blot. The result showed an asymmetric synergism between CymMV and ORSV, similar to that reported previously (Pai et al., 2020). CymMV infection suppressed ORSV accumulation during the entire infection period.
In contrast, CymMV accumulation was slightly higher in mix-infected leaves (Mix) than that in CymMV-infected leaves at 15 dpi ( Figure 2).
The PaAGO5b transcript accumulation level exhibited the most prominent increases in CymMV-and mix-infected leaves, by 6.7-and 14.5-fold, respectively ( Figure 3b). The increasing level of PaAGO5b The expression of PaAGO5 has been reported to be induced by the infection of CymMV or mixed infection of CymMV and ORSV (Pai et al., 2020); however, the levels of PaAGO5a, 5b, and 5c were not F I G U R E 3 The expression profile of PaAGOs in virus-infected leaves. The leaves of Phalaenopsis aphrodite subsp. formosana were inoculated with infectious clones of CymMV, ORSV, or both via agroinfiltration (indicated as CymMV, ORSV, and Mixed, respectively). The leaves were collected at 5 days postinoculation for total RNA extraction. The transcript accumulation levels were examined by quantitative reverse transcription PCR. The expression levels of each PaAGO transcript, presented as normalized fold changes relative to that from mock-inoculated leaves (EV), are shown. (a) PaAGO1a and PaAGO1b, (b) PaAGO4 and PaAGO6, (c) PaAGO7a and PaAGO7b, (d) PaAGO5a, PaAGO5b, and PaAGO5c, and (e) PaAGO10a and PaAGO10b. Values are means ± SD of three biological replicates. Norm. Exp, normalized expression level; *, ** and ***, significant difference at p < .05, p < .01, and p < .001 determined by Student's t test, respectively distinguished in the previous study. The present results further revealed that PaAGO5b may be the actual PaAGO5 that responded to CymMV infection.

| Overexpression of PaAGO5s confers resistance to CymMV and ORSV infections in P. aphrodite subsp. formosana
To further analyse the functions of PaAGO5s in antiviral defence, the coding sequences of all three PaAGO5s were individually cloned. Specific primers for cloning PaAGO5s were designed based on the sequences in the Orchidstra 2.0 database. PaAGO5 coding sequences were amplified using RT-PCR with respective primer pairs (Table 1) and the 5′ termini of the mRNAs were further examined by 5′ rapid amplification of cDNA ends (5′ RACE). The cDNAs of PaAGO5a (1,965 bp) and PaAGO5b (2,901 bp) share 100% and 99.9% identities with the sequences in the Orchidstra 2.0 database, respectively. The corresponding sequence for PaAGO5c in the database is only 975 nt long; however, our 5′ RACE analysis extended the 5′-terminal sequence and revealed that PaAGO5c mRNA is 2,040 nt in length. Protein sequence alignment showed that all PaAGO5s comprise the typical PAZ, MID, and PIWI domains with comparable sizes. The size of PaAGO5b is much larger than those of the other PaAGO5s, with an extended N-terminal domain ( Figure S2a). The characteristic DDH catalytic triad, key metal-coordinating residues involved in RNase H activity (Jullien et al., 2020), and RNA interacting region were conserved in all three PaAGO5s and shared a high sequence similarity ( Figure S2b). The above finding suggested that all PaAGO5s may be involved in the RNA silencing machineries.
To further investigate the antiviral activity, PaAGO5s were transiently overexpressed in virus-infected plants. The overexpression of PaAGO5s did not significantly affect the antiviral activity against CymMV or ORSV infections in N. benthamiana, possibly due to the incompatibility of exogenous PaAGO5s and the antiviral RNA silencing machinery of N. benthamiana. Therefore, the antiviral activities of PaAGO5s were directly examined in P. aphrodite subsp.

| Knockdown of PaAGO5s led to increased susceptibility to CymMV and ORSV infections in P. aphrodite subsp. formosana
To further corroborate the above observations, PaAGO5 transcripts were selectively silenced in P. aphrodite subsp. formosana, and the plants were then tested for susceptibility to CymMV or ORSV. Virus-induced gene silencing (VIGS) is one of the most efficient tools to knock down gene expression, as long as the plant can be inoculated by the chosen viral vector. As one of the most notorious viruses on orchids, CymMV has been used to establish an effective VIGS system for studies on flowering-related genes or plant defence response in Phalaenopsis orchid (Hsieh et al., 2013;Lu et al., 2012). However, CymMV-based VIGS vectors are not applicable in this study because CymMV is the target virus under investigation. We therefore developed an alternative VIGS system for this study. Several candidate VIGS vectors were tested for infectivity on P. aphrodite subsp. formosana. The coat proteins of tobacco mosaic virus (TMV) and potato virus X (PVX) were barely detectable on leaves that had been agroinfiltrated with pKTMV and pKPVXGFP (Huang et al., 2019) at 10 dpi (Figure 6a,b). In contrast, both BaMV-and foxtail mosaic virus (FoMV)-based VIGS vectors, pKBG (Prasanth et al., 2011) and pKFV, respectively, could infect P. aphrodite subsp. formosana, and the coat protein accumulation of FoMV was much higher than that of BaMV ( Figure 6c,d). Accordingly, pKFV was employed as a VIGS vector for the following experiments in this study. To evaluate the silencing efficiency of the FoMV-based VIGS vector, a fragment of the P. aphrodite phytoene desaturase gene (PaPDS) (Lu et al., 2007) was cloned into pKFV to generate pKFV-PaPDS. The infiltration of pKFV-PaPDS resulted in a 20% reduction of PaPDS transcript accumulation at 10 dpi, and the photobleaching phenotype on the inoculated leaves was observed at 60 dpi (Figure 6e). Although the photobleaching caused by FoMV-based VIGS was not as se-
formosana leaves through agroinfiltration. The leaves were collected and tested for PaAGO5 silencing efficiency at 10 dpi. The results showed that the transcripts of PaAGO5a, PaAGO5b, and PaAGO5c were decreased to 33%, 21%, and 30%, respectively, as compared to the empty vector-treated group (Figure 7b). Although

CymMV infections. Nevertheless, this study revealed that PaAGO5b
is an important antiviral protein that may serve as the frontline defence against CymMV and/or ORSV infections at the early stage, as PaAGO5b is the only significantly activated gene upon viral infections.

| Other members of the AGO1/5/10 clade are also reactive to viral infections
In addition to PaAGO5b, the expression of other genes in the AGO1/5/10 clade, PaAGO1a and PaAGO10s, were also found to be responsive to CymMV and/or ORSV infections in this study. AGO1 is widely and constitutively expressed in many model plants and is re- in P. aphrodite subsp. formosana. Another member of the AGO1/5/10 clade, AGO10, is also known to regulate several physiological characteristics in plants through competing with AGO1 for specific miR-NAs. For instance, AGO10 has been proposed to act as a decoy to sequestrate miR165/166 from loading into AGO1 and regulate the maintenance of the shoot apical meristem (Roodbarkelari et al., 2015;Zhu et al., 2011). Furthermore, NbAGO10 enhances the accumulation of BaMV by sequestering and degrading BaMV-derived vsiRNAs, preventing their incorporation into NbAGO1 (Huang et al., 2019).
However, in P. aphrodite subsp. formosana, PaAGO10a and PaAGO10b were both significantly decreased while singly or doubly infected with CymMV and/or ORSV (Figure 3e). Our observations suggest that CymMV and ORSV may have evolved the ability to suppress the antiviral defences mediated by PaAGO1s and/or PaAGO10s in P. aphrodite subsp. formosana, which in turn may have developed the ability to employ PaAGO5s as the primary antiviral components.

| The asymmetric synergism between CymMV and ORSV might be related to the differential responses of PaAGOs
Similar to previous reports (Pai et al., 2020), asymmetric synergism between CymMV and ORSV was also observed in this study, in which CymMV is the primary beneficiary in the synergistic relationship. The coat protein accumulation of CymMV was significantly higher under mixed-infection conditions at 15 dpi only; however, the accumulation of ORSV coat protein was significantly suppressed throughout the experiment (Figure 2). Although it could not be ruled out that CymMV might simply deprive ORSV of certain factors for replication or accumulation, it is likely that other viral or host factors are also involved in the asymmetric synergism.
Potexviruses, such as PVX and BaMV, encode TGBp1 proteins (p25 and p28, respectively) that are reported to be the important viral suppressors of RNA silencing (VSRs) for viral infections (Aguilar et al., 2015;Hsu et al., 2004). Moreover, p25 has been reported to inhibit the systemic movement of silencing signals in N. benthamiana (Voinnet et al., 2000). AGOs may also be involved in the process. AGO1 and AGO7 were found to be destabilized by PVX p25 (Brosseau & Moffett, 2015). AGO1, AGO6, AGO7, AGO9, and AGO10 exhibited lower expression levels with the coexpression of Plantago asiatica mosaic virus p25 (Brosseau et al., 2016). In contrast, members of the genus Tobamovirus, such as TMV and ORSV, also encode a well-studied VSR, p126. TMV p126 consists of three domains, methyltransferase, helicase, and nonconserved regions, and each of them has been reported to function independently as a silencing suppressor . A previous study showed In the present study, PaAGO4, PaAGO6, PaAGO7s, and PaAGO10s were all down-regulated in all virus-infected groups (Figure 3).
However, the accumulation of PaAGO5b was significantly elevated when P. aphrodite subsp. formosana plants were infected singly with CymMV or doubly with both viruses, but not ORSV alone.
Although there was no direct evidence demonstrating that CymMV TGBp1 and/or ORSV p126 were involved in the suppression of RNA

| Conclusion
This against CymMV and ORSV. The results provided deeper insights into the AGO-related antiviral mechanism in P. aphrodite subsp.
formosana, and suggested a promising strategy to develop resistance against viruses in orchid, either through breeding or transgenic approaches to enhance the expression of PaAGO5s. Future studies will focus on the regulation of PaAGO5 expressions in response to viral infections to decipher the interaction between viral factors and PaAGO5-associated resistance in P. aphrodite subsp. formosana.

| RNA extraction, RT-PCR, and quantitative PCR
Total RNA extraction and the elimination of DNA contamination were performed by using Direct-zol RNA MiniPrep (Zymo Research).
Following extraction, 2 µg of total RNA was used for first-strand cDNA synthesis with oligo(dT 18 ) primer and GoScript Reverse Transcriptase (Promega). The first-strand cDNA was then mixed with specific primer and KAPA SYBR FAST qPCR master mix (Kapa Biosystems). Quantitative real-time PCR was carried out using a TOptical Gradient 96 Real-Time PCR thermal cycler (Biometra) as described (Huang et al., 2019). The primer sequences are as listed in Table 1. Expression levels of target transcripts were normalized to the geometric mean of housekeeping gene, Actin, to control the variability and further analysed using the 2 −ΔΔCt method (Livak & Schmittgen, 2001). For the confirmation of reproducibility, three biological replicates of each essay were used for qPCR analysis, and three technical replicates were analysed for each biological replicate.

| Construction of PaAGO5 overexpression vector
The PaAGO5a, PaAGO5b, and PaAGO5c coding sequences (cds) were amplified by RT-PCR. The specific primer pairs were designed according to the cds of PaAGO5s in the Orchidstra 2.0 database as listed in Table 1 (Huang et al., 2020) by SbfI and XhoI digestion and then ligated with SbfI/XhoI-digested pKn vector to generate the pKFV vector. The fragment for gene silencing of P. aphrodite subsp. formosana endogenous phytoene desaturase (PaPDS) was amplified by PCR using PDS primers PDS-F and PDS-R (Lu et al., 2007). The fragment of PaPDS flanked by AgeI and NotI restriction sites was cloned into VIGS vector pKCy1 to generate the pKCy1-PDS vector.
The pKFV-PDS VIGS construct was created similarly using HpaIand MluI-digested PaPDS fragment and pKFV vector. The fragment used for silencing all three PaAGO5 genes, PaAGO5 u, in P. aphrodite subsp. formosana was flanked by HpaI and SpeI at the 5′ and 3′ ends, respectively. After PCR amplification, the product was digested by SpeI and self-ligated to generate an inverted repeat fragment of PaAGO5 u, designated PaAGO5uIR. PaPDS and PaAGO5uIR fragments were digested by HpaI and ligated with HpaI-digested pKFV to generate pKFV-PaPDS and pKFV-PaAGO5uIR, respectively.

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