Molecular Characterization and the Function of Argonaute3 in RNAi Pathway of Plutella xylostella

Argonaute (Ago) protein family plays a key role in the RNA interference (RNAi) process in different insects including Lepidopteran. However, the role of Ago proteins in the RNAi pathway of Plutella xylostella is still unknown. We cloned an Argonaute3 gene in P. xylostella (PxAgo3) with the complete coding sequence of 2832 bp. The encoded protein had 935 amino acids with an expected molecular weight of 108.9 kDa and an isoelectric point of 9.29. It contained a PAZ (PIWI/Argonaute/Zwile) domain and PIWI (P-element-induced whimpy testes) domain. PxAgo3 was classified into the Piwi subfamily of Ago proteins with a high similarity of 93.0% with Bombyx mori Ago3 (BmAgo3). The suppression of PxAgo3 by dsPxAgo3 was observed 3 h after treatment and was maintained until 24 h. Knockdown of PxAgo3 decreased the suppression level of PxActin by dsPxActin in P. xylostella cells, while overexpression of PxAgo3 increased the RNAi efficiency. Our results suggest that PxAgo3 play a key role in the double stranded RNA (dsRNA)-regulated RNAi pathway in P. xylostella.


Introduction
RNA interference (RNAi), also called double stranded RNA (dsRNA)-induced post-transcriptional gene silencing, is a cellular mechanism regulated by a unique group of RNA-binding proteins named Argonautes (Ago) [1,2]. Ago proteins bind to small administrative RNAs, such as small interfering RNA (siRNA), micro RNA (microRNA), and Piwi-interacting RNA (piRNA), to generate the RNA-induced silencing complex (RISC) [3]. Each Ago protein consists of two fundamental domains, PAZ (PIWI/Argonaute/Zwile) and PIWI (P-element-induced wimpy testes), in their structure [4]. Ago protein structures of eukaryotes and prokaryotes share a bilobed architectural structure containing four domains of N, PAZ, MID (middle) and, PIWI connected by two linkers [5]. The N domain is linked with a small RNA duplex loosening up during the RISC formation [6]. The PAZ domain contains

Identification and Sequence Analysis of PxAgo3 Gene
Using Ago3 CDS sequence from B. mori (BmAgo3, GenBank: AB372007.1), we identified Px011435 as the Ago3 coding sequence in P. xylostella genome. Using the specific primers of PxAgo3-F and PxAgo3-R (Table 1) based on Px011435, PxAgo3 was amplified through PCR and sequenced. The sequence was deposited in the GenBank with the accession number of MG778697. The sequence of PxAgo3 was of 2832 bp with the predicted protein of 943 aa ( Figure 1B). The cloned sequence contained two conserved regions with the PAZ domain of 420 bp in length and the Piwi domain of 1335 bp from 3 end to 5 end.
GenBank database sequence comparison showed that PxAgo3 was similar to B. mori Piwi-like Ago3 protein with the similarity of 49.3%. To analyze the functional sequence, the deduced amino acid sequence of PxAgo3 and some expressive Ago family protein sequences were aligned based on the conserved PIWI domain. The multiple-sequence alignment led to the identification of one 5 -phosphate-anchoring region and three catalytic residues (DDH positioned by red arrows, Figure 2) inside the PIWI domain. Thirty-one Ago proteins sequences from ten different species were collected through the NCBI website to construct the phylogenetic tree ( Table 2). The phylogenetic tree showed that the Ago proteins were divided into two subfamilies, Ago-and Piwi, with PxAgo3 in the Piwi subfamily located with BmAgo3 ( Figure 3).     Table 2. The bootstrap values of 1000 repeats are shown at the nodes.

PxAgo3 Expression in Response to dsRNA in P. xylostella Larvae
The expression of PxAgo3 was significantly more suppressed by dsPxAgo3 in the 3rd instar larvae of P. xylostella than by CK and dsEGFP. The suppression was first observed in 3 h after treatment and maintained up to 24 h ( Figure 4).  Table 2. The bootstrap values of 1000 repeats are shown at the nodes.

PxAgo3 Expression in Response to dsRNA in P. xylostella Larvae
The expression of PxAgo3 was significantly more suppressed by dsPxAgo3 in the 3rd instar larvae of P. xylostella than by CK and dsEGFP. The suppression was first observed in 3 h after treatment and maintained up to 24 h ( Figure 4).

PxAgo3 Suppression Decreased the RNAi Efficiency in P. xylostella Cells
We hypothesized that the knockdown of PxAgo3 by dsPxAgo3 would affect the suppression of endogenous gene PxActin by dsPxActin. Silencing of PxAgo3 by dsPxAgo3 led to a significant downregulation (F = 11.34, df1 = 4, df2 = 10, p = 0.0010) ( Figure 5A). For RNAi of RNAi, P. xylostella cells were exposed to dsPxAgo3 for 24 h, followed by exposure of these cells to dsPxActin for another 24 h.

PxAgo3 Suppression Decreased the RNAi Efficiency in P. xylostella Cells
We hypothesized that the knockdown of PxAgo3 by dsPxAgo3 would affect the suppression of endogenous gene PxActin by dsPxActin. Silencing of PxAgo3 by dsPxAgo3 led to a significant down-regulation (F = 11.34, df 1 = 4, df 2 = 10, p = 0.0010) ( Figure 5A). For RNAi of RNAi, P. xylostella cells were exposed to dsPxAgo3 for 24 h, followed by exposure of these cells to dsPxActin for another 24 h. PxActin expression was significantly suppressed by dsPxActin and dsPxActin+dsEGFP, but not by dsPxAgo3+dsPxActin (F = 9.91, df 1 = 4, df 2 = 10, p = 0.0017) ( Figure 5B). These results demonstrated that the dsRNA-mediated suppression of PxAgo3 reduced the RNAi efficiency in P. xylostella cells.

PxAgo3 Suppression Decreased the RNAi Efficiency in P. xylostella Cells
We hypothesized that the knockdown of PxAgo3 by dsPxAgo3 would affect the suppression of endogenous gene PxActin by dsPxActin. Silencing of PxAgo3 by dsPxAgo3 led to a significant downregulation (F = 11.34, df1 = 4, df2 = 10, p = 0.0010) ( Figure 5A). For RNAi of RNAi, P. xylostella cells were exposed to dsPxAgo3 for 24 h, followed by exposure of these cells to dsPxActin for another 24 h. PxActin expression was significantly suppressed by dsPxActin and dsPxActin+dsEGFP, but not by dsPxAgo3+dsPxActin (F = 9.91, df1 = 4, df2 = 10, p = 0.0017) ( Figure 5B). These results demonstrated that the dsRNA-mediated suppression of PxAgo3 reduced the RNAi efficiency in P. xylostella cells.

PxAgo3 Overexpression Increased the RNAi Efficiency in P. xylostella Cells
To overexpress PxAgo3 in P. xylostella cells, the expression construct was inserted into the PIZT/V5 vector and transfected into P. xylostella cells. The success of transfection was verified by the enhanced green fluorescence protein (EGFP) expression with the green fluorescence observed under the florescence microscope ( Figure 6A,B) and by PCR using vector-based primers ( Figure 6C). The expression level of PxAgo3 in P. xylostella cells significantly increased in transfected P. xylostella cells (F = 10.45, df1 = 2, df2 = 6, p = 0.0111) ( Figure 6D). To observe the RNAi efficiency in PxAgo3overexpressed cells, PxActin was targeted to suppress in PxAgo3-overexpressed cells. The PxActin

PxAgo3 Overexpression Increased the RNAi Efficiency in P. xylostella Cells
To overexpress PxAgo3 in P. xylostella cells, the expression construct was inserted into the PIZT/V5 vector and transfected into P. xylostella cells. The success of transfection was verified by the enhanced green fluorescence protein (EGFP) expression with the green fluorescence observed under the florescence microscope ( Figure 6A,B) and by PCR using vector-based primers ( Figure 6C). The expression level of PxAgo3 in P. xylostella cells significantly increased in transfected P. xylostella cells (F = 10.45, df 1 = 2, df 2 = 6, p = 0.0111) ( Figure 6D). To observe the RNAi efficiency in PxAgo3-overexpressed cells, PxActin was targeted to suppress in PxAgo3-overexpressed cells. The PxActin expression was affected by the treatments (F = 45.95, df 1 = 4, df 2 = 10, p < 0.0001). PxActin expression was reduced by dsPxActin in normal cells and the suppression level was increased in PxAgo3-overexpressing cells ( Figure 6E). These results suggested that overexpression of PxAgo3 increased the dsRNA-regulated RNAi efficiency in P. xylostella cells. expression was affected by the treatments (F = 45.95, df1 = 4, df2 = 10, p < 0.0001). PxActin expression was reduced by dsPxActin in normal cells and the suppression level was increased in PxAgo3overexpressing cells ( Figure 6E). These results suggested that overexpression of PxAgo3 increased the dsRNA-regulated RNAi efficiency in P. xylostella cells.

Discussion
In this study, we have cloned and identified a new Ago gene from P. xylostella, PxAgo3. The amino acid sequence of PxAgo3 included a PAZ domain and a PIWI domain. PAZ and PIWI are similar in different species, such as P. monodon [9], B. mori [16], D. melanogaster [15]. The PAZ domain is a particular nucleic acid binding site, which plays a significant role in RNAi mechanism [7]. Ago3 is physically bound with nucleic acid through the PAZ domain, while the PIWI domain interacts with the 5′ end of the guide RNA in Archaeoglobus fulgidus [30]. The three catalytic residues (DDH) within the PIWI domain of Ago3 form a cleavage triad in DmAgo3 protein [31], in BmAgo3 protein [32], and in PmAgo3 protein [9], and was also observed in PxAgo3.

Discussion
In this study, we have cloned and identified a new Ago gene from P. xylostella, PxAgo3. The amino acid sequence of PxAgo3 included a PAZ domain and a PIWI domain. PAZ and PIWI are similar in different species, such as P. monodon [9], B. mori [16], D. melanogaster [15]. The PAZ domain is a particular nucleic acid binding site, which plays a significant role in RNAi mechanism [7]. Ago3 is physically bound with nucleic acid through the PAZ domain, while the PIWI domain interacts with the 5 end of the guide RNA in Archaeoglobus fulgidus [30]. The three catalytic residues (DDH) within the PIWI domain of Ago3 form a cleavage triad in DmAgo3 protein [31], in BmAgo3 protein [32], and in PmAgo3 protein [9], and was also observed in PxAgo3.
The silencing of PxAgo3 occurred 3 h after dsRNA treatment and was maintained for 24 h. Ago3 knockdown is observed 24 h after introduction of dsRNA in mammalian cells [38] as well as A. aegypti, and A. albopictus cells [39]. In Culex quinquefasciatus, Ago3 suppression is only observed 48 h after treatment [40]. Therefore, it is suggested that Ago3 can be effectively suppressed by dsRNA in animals.
PxAgo3 knockdown reduced RNAi efficiency in P. xylostella cells. Ago3 is involved in dsRNA-regulated RNAi phenomenon [22] and reduces RNAi response in D. melanogaster [41] and B. mori [42]. Ago2 has been considered the only cleavage component in siRNA-regulated RNAi process in insects [42,43]. Our results, however, suggest that Ago3 may be involved in the siRNA-regulated RNAi pathway, which is known to be associated with the piRNA pathway. The overexpression of PxAgo3 also enhanced the RNAi response. The overexpression of Ago3 in Lepidopteran cell lines [26], C. elegans [14], as well as HEK293 and HeLa cells [21] can increase the RNAi efficiency. It is possible that PxAgo3 overexpression may facilitate the dsRNA-mediated RNAi in P. xylostella.
In conclusion, we sequenced PxAgo3 and identified PxAgo3 as a Piwi subfamily member containing the cleavage motif of DDH in the PIWI domain. We discovered that Ago3 is involved in the siRNA-mediated pathway in P. xylostella by decreasing PxActin suppression by dsPxActin through knockdown of PxAgo3 and enhancing PxActin suppression by dsPxActin through overexpression of PxAgo3 in P. xylostella cells.

P. xylostella Fuzhou-S Rearing and Cell Culture
The P. xylostella individuals used in this study came from an insecticide-susceptible strain previously used for P. xylostella genomic sequencing [44]. P. xylostella larvae were reared on radish seedlings at 25 ± 1 • C, 65 ± 5% RH and 16 h L: 8 h D. A stable P. xylostella cell line was used, which was established from embryonic tissues of P. xylostella [45]. Several generations of P. xylostella cells were maintained at 27 • C in Grace's medium (Invitrogen, Waltham, MA, USA) supplemented with 10% of fetal bovine serum (FBS).

Total RNAs Purification and cDNA Preparation
Total RNAs were purified from four 3rd instar larvae of P. xylostella using the TRIzol-Reagent (Invitrogen). The quantity of RNAs were measured using a Nanodrop ND-1000 spectrophotometer (Invitrogen), and the RNA quality was verified through 1% gel electrophoresis. Total RNAs were diluted with nuclease-free water (Promega, Madison, WI, USA) and maintained at a concentration of 500 ng/µL. Furthermore, 1 µL of purified RNA was used as the template for cDNA preparation by using the GoTaq ® 2-Step RT-qPCR System (Promega).

Molecular Cloning and Expression Vector Construction of PxAgo3
For PxAgo3 cloning, a specific pair of primers (PxAgo3-F and PxAgo3-R) ( Table 1) were designed using the Primer Premier 5 according to the gene sequence of PxAgo3 (gene ID: Px011435) from P. xylostella genome (Available online: http://iae.fafu.edu.cn/DBM/index.php). The Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) was used to conduct PCR following the manufacture protocol and conditions: denaturation at 95 • C for 3 min, amplification by 30 cycles at 95 • C for 15 s, 56 • C for 15 s, and 72 • C for 3 min, the last expansion at 72 • C for 10 min and then storage at 4 • C. The PCR product was extracted from the gel by using the QIAquick Gel extraction kit (Qiagen, Venlo, The Netherlands) according to the manufacturer protocol. The extracted PCR product and PJET vector were ligated using T4 DNA ligase enzyme (Promega) according to the ClonJET PCR Cloning kit (Invitrogen) and incubated at 25 • C for 1 h. The ligated sample was mixed with 50 µL high efficiency competent cells of DH-5α (Tiangen, Beijing, China) and incubated on ice for 30 min. The sample was cultured in the selection media to obtain white colonies. The colony PCR was done using the Go Taq ® Green Master Mix enzyme (Promega) with the vector-based primers ( Table 1). The PCR product was verified through 1% gel electrophoresis following the procedure previously described. Eight white colonies were checked and one verified positive white colony was cultured.
An amount of 500 µL of the cultured solution was sent to General Biosystems (China) for sequencing. The protein sequence of PxAgo3 was blasted in the National Center for Biotechnology Information (NCBI) (Available online: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGETYPE= BlastSearch&LINK_LOC=blasthome) to verify its identity and submitted to GenBank (Available online: https://submit.ncbi.nlm.nih.gov/subs/genbank/) for accession number.
To construct the PxAgo3 expression vector (PIZT/V5-His+PxAgo3), the PCR product and PIZT/V5-His were individually double-digested at 37 • C using two restriction enzymes of FastDigest KpnI and EcoRI (Invitrogen). Both double digested products (PCR product and PIZT/V5-His) were ligated and transfected into competent cells and checked by colony PCR and 1% gel electrophoresis (as previously described). A positive white colony cultured solution of PxAgo3 expression vector was extracted using the Hispeed Plasmid Midi kit (Qiagen).

Phylogenetic Tree and Sequence Analysis
Ago protein sequences were downloaded from the GenBank database of NCBI (Available online: https://www.ncbi.nlm.nih.gov/) and are listed in Table 2. A multiple-sequence alignment was performed based on the PIWI domain using the ClustalW software. The phylogenetic tree was constructed using MEGA 6.0 based on the neighbor-joining method with a bootstrap value of 1000 replicates. The isoelectric point and molecular weight of PxAgo3 were identified using the Expasy website (Available online: http://www.expasy.org).

Double-Stranded RNA (dsRNA) Preparation
The following gene-specific sequences were used as the dsRNA templates: 389-bp fragment targeting PxAgo3, 461-bp fragment targeting PxActin, and 457-bp fragment targeting EGFP. DsRNAs were prepared using the Ambion MEGAscript RNAi kit (Invitrogen). The primers listed in Table 1 were used to amplify these fragments with the T7-polymerase promoter (TAATACGACTCACTATAGGG) sequence. The reaction mixture was incubated at 37 • C for 3 h, heat shocked at 75 • C for 5 min and then kept at room temperature for 1 h for the annealing reaction. Subsequently, dsRNAs were treated with DNase I to remove the template DNA, purified with the phenol/chloroform method and finally diluted in nuclease-free water. The dsRNA quality and quantity were measured following the procedure previously described.

DsRNA Injection and Transfection into P. xylostella Larvae and Cells
For dsRNA injection, 3rd instar larvae of P. xylostella were selected and borosilicate micro-capillaries (Glass Replacement 1.1) were pulled using heater setting 64 in PC-10 (Narishige, Tokyo, Japan). We injected 10 µL dsRNAs (1 µg) of PxAgo3, PxActin and EGFP (control) by using micro-injection world precision instruments (Sarasota, FL, USA) into the hemolymph of 3rd instar larvae (n = 60) individually. After injection, larvae were transferred into separate plastic rearing box and fed with fresh radish leaves. Purified dsRNAs were transfected into the P. xylostella cells using the Cellfectin ® II reagent (Promega) according to the manufacturer's instructions.

RNAi of RNAi Assay in P. xylostella Cells
For RNAi of RNAi assay, one day before transfection of dsRNAs into cells, P. xylostella cells were cultured in 2 mL of Sf-900 III serum free media (SFM) (Promega) with 10% fetal bovine serum (FBS) (HyClone, Utah, United States) and containing 1% anti-toxin antimycotic (pH 6.2) at a concentration of 6 × 10 5 cells/mL, in 6-well plates (Corning Incorporated, New York, NY, USA). Cells were incubated at 27 • C overnight or until 50-60% of the cells converged. We transfected two types of dsRNA into P. xylostella cells in order. Firstly, 1 µg of dsPxAgo3 was transfected into P. xylostella cells by following the Cellfectin ® (Gibco Invitrogen, Waltham, MA, USA) manufacturer's instructions. The treated P. xylostella cells were incubated at 27 • C for 24 h in a closed chamber to prevent environmental contamination. Secondly, 1 µg of dsPxActin was transfected using the Cellfectin reagent (described as before) into the same P. xylostella cells 24 h after the first transfection of dsPxAgo3. The transfection of dsEGFP was used as a control. The PxAgo3 expression level was analyzed by using RT-qPCR. Similarly, the overexpression of PxAgo3 was measured after transfection of PIZT/V5-His+PxAgo3 into P. xylostella cells by RT-qPCR. The presence of vectors was examined through green fluorescence by using a fluorescence microscope Olympus IX51 (Olympus Corporation, Tokyo, Japan).
The cells were collected 48 h post transfection in 1.5 mL tubes (Axygen Incorporation, Tewksbury, MA, USA) and centrifuged at 1500× g for 5 min at 4 • C. Total RNA extraction, quality and quantity verifications, and cDNA preparation were performed as previously described. PxAgo3 and PxActin expression levels were monitored through RT-qPCR using the primers listed in Table 1. All procedures were done with three biological replicates.

RT-qPCR Analysis
RT-qPCR was performed using the GoTaq ® qPCR master mix (Promega). RT-qPCR was performed according to the following conditions: at 95 • C for 3 min for denaturation, followed by 40 cycles at 95 • C for 5 s and 60 • C for 20 s. The degree of amplification was determined at the end of each cycle by fluorescence intensity measurements. Quantitative mRNA measurements were done in triplicate and standardized to an internal control of ribosomal protein 32 (RL-32) (reference gene). All of the RT-qPCR primers are listed in Table 1.

Statistical Analysis
One-way analysis of variance (ANOVA) was done using the GraphPad (version 5.01) (GraphPad Software, La Jolla, San Diego, CA, USA), and then differences between treatments were determined using Tukey test at p < 0.05.