Ubiquitin-Conjugating Enzyme E2 E Inhibits the Accumulation of Rice Stripe Virus in Laodelphax striatellus (Fallén)

The ubiquitin–proteasome system (UPS) is an essential protagonist in host–pathogen interactions. Among the three classes of enzymes in the UPS, ubiquitin-conjugating enzyme E2 plays a dual role in viral pathogenesis; however, the role of insect E2s in interactions with plant viruses is unclear. Twenty E2-encoding genes in Laodelphax striatellus, the small brown planthopper, were identified and classified into 17 groups by transcriptomic and phylogenetic analysis. Full-length cDNAs of four LstrE2s (LstrE2 A/E/G2/H) were obtained by rapid-amplification of cDNA ends (RACE-PCR) analysis. Expression of the four LstrE2s showed tissue- and development-specific patterns. RT-qPCR analyses revealed that Rice stripe viruse (RSV) infection increased the level of LstrE2 A/E/G2/H. Further study indicated that repression of LstrE2 E via RNAi caused significant increases in the expression of RSV coat protein mRNA and protein levels. These findings suggest that LstrE2 E inhibits RSV accumulation in the planthopper body. Understanding the function of LstrE2 E in RSV accumulation may ultimately result in the development of novel antiviral strategies.


Introduction
Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins are highly-conserved molecules that generally contain around 100 amino acids that can be covalently attached to protein substrates through a versatile and reversible modification known as ubiquitination [1][2][3]. Ubiquitination is a dynamic posttranslational modification that contributes to virtually all aspects of cell biology including cellular proliferation, DNA repair, apoptosis, and antigen processing [4][5][6]. Protein ubiquitination is a three-step enzymatic process that requires a series of enzymes, including ubiquitin-activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). Generally, the process starts with the activation of Ub or Ubl by E1, followed by E2 and E3, which transfer and conjugate the activated Ub or Ubl to a lysine residue within the protein substrate [2]. The most common outcome of ubiquitination is protein substrate recognition and degradation by the 26S proteasome [7]. Among the ubiquitin enzymes, E2 carries the activated Ub/Ubl from the Ub/Ubl-E1 thioester to E3 or occasionally completes the conjugation of Ub/Ubl to target proteins in the absence of E3 [4,8]. During this modification, E2 determines the processivity and topology of polyubiquitin chain formation, which ultimately regulates the fate, function, and interaction of target proteins [9,10]. Most insect species contain 20 with a monoclonal anti-RSV CP antibody [39]. Highly viruliferous colonies were retained and used in subsequent studies.

Cloning and Structural Analysis of LstrE2 Genes
Total RNA was isolated from 20 planthopper adults, using TRIzol reagent as recommended (Invitrogen), and RNA quality and concentration were determined by spectrophotometry (NanoDrop, Thermo Scientific, Waltham, MA, USA). RNA (500 ng) was used for reverse transcription in a 10 µl reaction volume with the PrimeScript™ RT reagent kit and gDNA Eraser as recommended (Takara, Dalian, China). Based on the mRNA sequences of LstrE2s obtained from transcriptomes, 5 and 3 rapid-amplification of cDNA ends (RACE) were conducted to obtain full-length LstrE2 transcripts (Takara). Predicted LstrE2 proteins were subjected to Blast analysis using DNAman software (LynnonBiosoft, Los Angeles, CA, USA), and domains of predicted proteins were deduced using SMART (http://smart.embl-heidelberg.de/) [43].

Real-Time RT-qPCR
To measure LstrE2 expression and RSV copy number equivalents in small brown planthopper, total RNA was isolated from 20 intact bodies, 50 midgut/ovaries, and 100 salivary glands of adults and nymphs (female/male ratio = 1:1) using the TRIzol Total RNA Isolation Kit (Takara, Dalian, China). Total RNA concentrations were quantified, and first-strand cDNA was synthesized as described above. The primers (Table S1) used for detecting RSV copy number equivalents were designed based on RSV CP-specific nucleotide sequences. Similarly, LstrE2s and LstrActin (control) primers (Table S2) were designed based on LstrE2s and LstrActin sequences, respectively. RT-qPCR was conducted using a CFX96™ Real-Time PCR Detection System using reagents and parameters described previously [40]. Relative expression levels for triplicate samples were calculated using the ∆∆Ct method, and expression levels of target genes were normalized to LstrActin. Three technical repeats were performed for each of the three biological replicates.

RNA Interference (RNAi)
The coding sequences of LstrE2s and GFP were cloned into pMD19-T (Takara, Japan). The primers for dsGFP and dsLstrE2 amplification are listed in Table S2. Using the cDNA templates obtained above, dsRNAs were synthesized using the T7 RiboMAX™ Express RNAi System kit as recommended by the manufacturer (Promega, USA). Third-instar naïve nymphs were microinjected by dsLstrE2s (23 nL, 2.5 µg/µL) or dsGFP (23 nL, 2.5 µg/µL) using an UMP3-2 UltraMicroPump (UMP3) and a SYS-Micro4 Controller (WPI, Sarasota, FL, USA) [39]. Following microinjection, nymphs were transferred and maintained on healthy rice seedlings until analyzed by immunofluorescence, RT-qPCR, or Western blot analysis. The impact of dsRNA on the expression of LstrE2s was evaluated by RT-qPCR.

Immunofluorescence Microscopy
Ten or more planthopper adults were maintained on rice plants for seven days after RNAi treatment and then dissected to obtain midgut and salivary glands. The dissected samples were fixed with 4% paraformaldehyde for 1 h. Samples were then blocked using 10% fetal bovine serum at ambient temperature for 2 h. Samples were incubated for 16 h at 4 • C with preimmune serum and anti-RSV CP antibody (1:500 dilution) before incubation with Alexa Fluor 488-labeled secondary goat anti-rabbit IgG. Salivary glands were then washed three times in: Phosphate Buffered Saline (PBS) and stained with 100 nM 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) for 2 min at room temperature. Fluorescence was observed with a Leica TCS SP8 STED confocal microscope (Leica, Wetzlar, HE, Germany).

Yeast Two-Hybrid Assays
Yeast two-hybrid assays were conducted using protocols supplied with the Yeastmaker™ Yeast Transformation System 2 (Takara-Clontech, USA). Briefly, the cDNA library of RSV was cloned as prey in plasmid vector pGADT7 using the Easy Clone cDNA library construction kit (Dualsystems Biotech); full-length LstrE2 E was cloned as bait in pGBKT7. Positive clones were selected on SD quadruple-dropout (QDO) medium (SD/-Ade/-His/-Leu/-Trp). To distinguish positive from false-positive interactions, we co-transformed BD-53 and AD-T, AD-LstrE2 E and BD-LstrE3, AD-LstrVg and BD-RSV CP into yeast strain Y2HGold as positive controls, respectively. ß-galactosidase activity was detected with the HTX Kit (Dualsystems Biotech).

GST Pull-Down Assay
LstrE2 E cDNA fragments were amplified and cloned into pGEX-3X as glutathione-S-transferase (GST) translational fusions. Recombinant proteins were produced in Escherichia coli strain BL21 and purified. For pull-down assays, viruliferous small brown planthopper extract (1 mg), immobilized glutathione-sepharose beads (200 µL), and GST-LsTUB protein (500 µg) were added to 1 mL of pull-down buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 1 mM Phenylmethanesulfonyl fluoride (PMSF), 1% protease inhibitor cocktail [pH 8.0]) and then incubated at 4 • C for 16 h. Similarly, insect extracts were incubated with GST protein as a negative control. Beads were washed four times with pull-down buffer, and retained proteins were released by adding 2× loading buffer and incubating for 5 min at 95 • C. Proteins were then separated by SDS-PAGE and detected using anti-GST (Cusabio, China) and anti-LstrE3 antibodies (prepared in our laboratory).

Identification and Classification of LstrE2s
The transcriptomes of intact small brown planthopper bodies were sequenced using the Illumina HiSeq™ 2000 platform. More than 42.94 million clean reads were obtained from each transcriptome and over 60.9% mapped to the small brown planthopper genome (Table S2). The mapped genes annotated as candidates of ubiquitin-conjugating enzyme (E2) were screened with a BLASTx algorithm-based search using known E2 genes form Drosophila melanogaster and other insect species. Twenty E2 genes were identified in small brown planthopper, which included the ubiquitin-conjugating and ubiquitin-like conjugating enzymes (Table 1). A phylogenetic tree was constructed for LstrE2s and proteins containing ubiquitin-conjugating (UBC) domains from six insect species in orders Polyneoptera, Paraneoptera, Coleoptera, Hymenoptera, Diptera, and Lepidoptera ( Figure 1). Almost all insect E2s were grouped into 19 clades comprised of E2 W, E2 Q, lessright (Lwr), E2 G, E2 R, E2 J2, BIR-repeat containing ubiquitin-conjugating enzyme (Bruce), E2 O, E2 M, E2 L3, E2 S, E2 H, E2 A, vihar (Vih), E2 E, effete (Eff), bendless (Ben), E2 T, and UBC4; this assignment is consistent with the nomenclature of E2 in human cells [44]. The 20 LstrE2s were distributed among these 17 groups, which suggested shared functions with other insect E2s.

Identification and Classification of LstrE2s
The transcriptomes of intact small brown planthopper bodies were sequenced using the Illumina HiSeq TM 2000 platform. More than 42.94 million clean reads were obtained from each transcriptome and over 60.9% mapped to the small brown planthopper genome (Table S2). The mapped genes annotated as candidates of ubiquitin-conjugating enzyme (E2) were screened with a BLASTx algorithm-based search using known E2 genes form Drosophila melanogaster and other insect species. Twenty E2 genes were identified in small brown planthopper, which included the ubiquitinconjugating and ubiquitin-like conjugating enzymes (Table 1). A phylogenetic tree was constructed for LstrE2s and proteins containing ubiquitin-conjugating (UBC) domains from six insect species in orders Polyneoptera, Paraneoptera, Coleoptera, Hymenoptera, Diptera, and Lepidoptera ( Figure 1). Almost all insect E2s were grouped into 19 clades comprised of E2 W, E2 Q, lessright (Lwr), E2 G, E2 R, E2 J2, BIR-repeat containing ubiquitin-conjugating enzyme (Bruce), E2 O, E2 M, E2 L3, E2 S, E2 H, E2 A, vihar (Vih), E2 E, effete (Eff), bendless (Ben), E2 T, and UBC4; this assignment is consistent with the nomenclature of E2 in human cells [44]. The 20 LstrE2s were distributed among these 17 groups, which suggested shared functions with other insect E2s.

Cloning and Sequence Analysis of Four LstrE2s
Full-length cDNA sequences of four LstrE2s were cloned from planthopper adults using conserved LstrE2 sequences as an in-silico probe. LstrE2 A (GenBank accession no. MT334578) is a 1089-bp cDNA that encodes a putative protein of 151 amino acids with a theoretical molecular weight (MW) and isoelectric point ( Figure 2A). Phylogenetic analysis revealed that the four LstrE2s had high sequence identity with other insect E2s deposited in the NCBI database and were very closely related to E2s in Nilaparvata lugens ( Figure 2B).

Cloning and Sequence Analysis of Four LstrE2s
Full-length cDNA sequences of four LstrE2s were cloned from planthopper adults using conserved LstrE2 sequences as an in-silico probe.  (Figure 2A). Phylogenetic analysis revealed that the four LstrE2s had high sequence identity with other insect E2s deposited in the NCBI database and were very closely related to E2s in Nilaparvata lugens ( Figure 2B).

Expression Analysis of the Four LstrE2s
RT-qPCR was used to evaluate LstrE2 A/E/G2/H mRNA expression in different tissues and developmental stages of small brown planthopper. Three LstrE2s (LstrE2 A, E, H) were more highly expressed in midgut than in salivary glands or ovaries ( Figure 3A,B,D), while the LstrE2 G2 was most highly expressed in ovaries ( Figure 3C). The four LstrE2 expression profiles in the five developmental stages were very similar. The highest transcription level was detected in planthopper adults sampled three days after molting (Figure 4).

Expression Analysis of the Four LstrE2s
RT-qPCR was used to evaluate LstrE2 A/E/G2/H mRNA expression in different tissues and developmental stages of small brown planthopper. Three LstrE2s (LstrE2 A, E, H) were more highly expressed in midgut than in salivary glands or ovaries ( Figure 3A,B,D), while the LstrE2 G2 was most highly expressed in ovaries ( Figure 3C). The four LstrE2 expression profiles in the five developmental stages were very similar. The highest transcription level was detected in planthopper adults sampled three days after molting (Figure 4).  RT-qPCR analysis of (A) LstrE2 A, (B) LstrE2 E, (C) LstrE2 G2, and (D) LstrE2 H expression in midguts, salivary glands, and ovaries of adults. In total, 50 midguts, 100 salivary glands, and 50 ovaries were considered to be a single replicate, and each treatment contained three replicates. All expressions are relative to first column in panel A. Bars labeled with different letters indicate significant differences in expression levels using RT-qPCR (p < 0.05). ovaries were considered to be a single replicate, and each treatment contained three replicates. All expressions are relative to first column in panel A. Bars labeled with different letters indicate significant differences in expression levels using RT-qPCR (p < 0.05).

Figure 4. The expression of LstrE2 A/E/G2/H at different developmental stages of small brown planthopper.
RT-qPCR analysis of (A) LstrE2 A, (B) LstrE2 E, (C) LstrE2 G2, and (D) LstrE2 H expression in 20 insects at 3rd instar, 5th instar, 1 day after molting, 3 days after molting, and 5 days after molting. In total, 20 insects were considered to be a single replicate, and each treatment contained three replicates. All expressions are relative to first column in the panel A. Bars labeled with different letters indicate significant differences in expression (p < 0.05).

Rice Stripe Virus Increases LstrE2s Expression in Small Brown Planthopper Adults
RT-qPCR analyses were conducted to evaluate expression levels of the four LstrE2s in viruliferous and virus-naïve planthopper adults. The mRNA expression levels of the four LstrE2s were significantly upregulated in viruliferous planthopper adults ( Figure 5); for example, the expression of LstrE2 A, LstrE2 E, LstrE2 G2, and LstrE2 H increased by 54.2%, 220.7%, 100.7%, and 150.5%, respectively, when compared to viruliferous-naïve planthopper adults ( Figure 5A-D). RT-qPCR analysis of (A) LstrE2 A, (B) LstrE2 E, (C) LstrE2 G2, and (D) LstrE2 H expression in 20 insects at 3rd instar, 5th instar, 1 day after molting, 3 days after molting, and 5 days after molting. In total, 20 insects were considered to be a single replicate, and each treatment contained three replicates. All expressions are relative to first column in the panel A. Bars labeled with different letters indicate significant differences in expression (p < 0.05).

Rice Stripe Virus Increases LstrE2s Expression in Small Brown Planthopper Adults
RT-qPCR analyses were conducted to evaluate expression levels of the four LstrE2s in viruliferous and virus-naïve planthopper adults. The mRNA expression levels of the four LstrE2s were significantly upregulated in viruliferous planthopper adults ( Figure 5); for example, the expression of LstrE2 A, LstrE2 E, LstrE2 G2, and LstrE2 H increased by 54.2%, 220.7%, 100.7%, and 150.5%, respectively, when compared to viruliferous-naïve planthopper adults ( Figure 5A-D).

Figure 5. LstrE2 A/E/G2/H expression in virus-naïve and viruliferous small brown planthopper.
RT-qPCR analysis of (A) LstrE2 A, (B) LstrE2 E, (C) LstrE2 G2, and (D) LstrE2 H expression in naïve and viruliferous adults. In total, 20 insects were considered to be a single replicate, and each treatment contained three replicates. All expressions are relative to first column in the panel A. Means ± S.E; ttest analysis, ** p < 0.01.

Repression of LstrE2 E via RNAi Increases RSV Load in Small Brown Planthopper
To further explore the function of the four LstrE2s in RSV infection, 3rd instar viruliferous planthopper nymphs were microinjected with 0.5 mg/mL dsRNAs derived from GFP (dsGFP) or LstrE2 A/E/G2/H (dsLstrE2 A/E/G2/H). At seven days post-dsRNA treatment, RT-qPCR analyses showed that LstrE2 mRNA expression levels in the corresponding dsLstrE2-treated planthoppers were significantly reduced by 61.4-90.4% compared to controls (dsGFP-treated planthoppers) ( Figure  6A). These results indicated that RNAi-mediated knockdown of the four LstrE2s was highly effective. RT-qPCR indicated that only the dsLstrE2 E treatment caused an increase in RSV copy number equivalents of viruliferous planthopper ( Figure 6B). Furthermore, RSV load and distribution were examined in planthopper whole bodies and different tissues via RT-qPCR, Western blotting, and confocal microscopy. RSV copy number equivalents were elevated in the 12 dsLstrE2 E-treated planthoppers ( Figure 7A), which agrees with results obtained with mixed samples ( Figure 6B). Immunoblotting indicated that the trend of RSV CP protein production was consistent with changes in mRNA expression ( Figure 7B). Confocal microscopy indicated that RSV particles were also present in midgut, salivary glands, and ovaries of dsLstrE2 E-treated planthoppers; furthermore, RSV

Figure 5. LstrE2 A/E/G2/H expression in virus-naïve and viruliferous small brown planthopper.
RT-qPCR analysis of (A) LstrE2 A, (B) LstrE2 E, (C) LstrE2 G2, and (D) LstrE2 H expression in naïve and viruliferous adults. In total, 20 insects were considered to be a single replicate, and each treatment contained three replicates. All expressions are relative to first column in the panel A. Means ± S.E; t-test analysis, ** p < 0.01.

Repression of LstrE2 E via RNAi Increases RSV Load in Small Brown Planthopper
To further explore the function of the four LstrE2s in RSV infection, 3rd instar viruliferous planthopper nymphs were microinjected with 0.5 mg/mL dsRNAs derived from GFP (dsGFP) or LstrE2 A/E/G2/H (dsLstrE2 A/E/G2/H). At seven days post-dsRNA treatment, RT-qPCR analyses showed that LstrE2 mRNA expression levels in the corresponding dsLstrE2-treated planthoppers were significantly reduced by 61.4-90.4% compared to controls (dsGFP-treated planthoppers) ( Figure 6A). These results indicated that RNAi-mediated knockdown of the four LstrE2s was highly effective. RT-qPCR indicated that only the dsLstrE2 E treatment caused an increase in RSV copy number equivalents of viruliferous planthopper ( Figure 6B). Furthermore, RSV load and distribution were examined in planthopper whole bodies and different tissues via RT-qPCR, Western blotting, and confocal microscopy. RSV copy number equivalents were elevated in the 12 dsLstrE2 E-treated planthoppers ( Figure 7A), which agrees with results obtained with mixed samples ( Figure 6B). Immunoblotting indicated that the trend of RSV CP protein production was consistent with changes in mRNA expression ( Figure 7B). Confocal microscopy indicated that RSV particles were also present in midgut, salivary glands, and ovaries of dsLstrE2 E-treated planthoppers; furthermore, RSV immunofluorescence was more intense in dsLstrE2 E-treated than dsGFP-treated planthopper ( Figure 7C, Figure S1). These results indicated that repression of LstrE2 E facilitated RSV accumulation in the planthopper body.
Viruses 2020, 12, x FOR PEER REVIEW 10 of 16 immunofluorescence was more intense in dsLstrE2 E-treated than dsGFP-treated planthopper ( Figure  7C, Figure S1). These results indicated that repression of LstrE2 E facilitated RSV accumulation in the planthopper body.  In total, 20 treated nymphs were considered to be a single replicate, and each treatment contained three replicates. Means ± S.E. One-way ANOVO analysis: different letters above bars (a,b,c) indicate significant differences of expression level between treatments. replicates. Means ± S.E. One-way ANOVO analysis: different letters above bars (a,b,c) indicate significant differences of expression level between treatments..

Figure 7. The effects of LstrE2 E knockdown on RSV accumulation and CP levels in viruliferous small brown planthopper. (A) RT-PCR analysis of RSV copy number equivalents in single dsLstrE2
E-and dsGFP-treated planthopper. Single insect was considered to be a single replicate, and each treatment was replicated twelve times; means ± S.E. (B) Western blot analysis of RSV CP in dsLstrE2 E-and dsGFP-treated adults. In total, 20 treated adults were mixed and used for protein extraction. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control. (C) Immunofluorescence in midgut, salivary glands and ovaries of viruliferous planthopper treated with dsLstrE2 E and dsGFP. Anti-RSV CP (Alexa Fluor 488, green) and DAPI (blue) were used as fluorescent probes. Each treatment was replicated five times. Abbreviations: mg, midgut; sg, salivary glands; o, ovary; vm, visceral muscle; me, midgut epithelium; psg, principal salivary glands; gr, germarium. Bar = 50 μm.

LstrE2 E Does Not Directly Interact with RSV Proteins
Considering that LstrE2 E may mediate RSV load by binding viral proteins, we used yeast twohybrid assays to evaluate whether LstrE2 E interacts with seven known RSV proteins (CP, SP, NS2, NS3, NSvc2, NSvc4, and RdRp). Full-length LstrE2 E was used as bait, and each of the six intact proteins (CP, SP, NS2, NS3, NSvc2, and NSvc4) and five truncated RdRp mutants were used as prey. All yeast strains failed to grow on synthetic dextrose dropout medium (Figure 8, Figure S2). This result suggested that LstrE2 E does not directly interact with RSV proteins.

Figure 7. The effects of LstrE2 E knockdown on RSV accumulation and CP levels in viruliferous small brown planthopper. (A) RT-PCR analysis of RSV copy number equivalents in single dsLstrE2
Eand dsGFP-treated planthopper. Single insect was considered to be a single replicate, and each treatment was replicated twelve times; means ± S.E. (B) Western blot analysis of RSV CP in dsLstrE2 Eand dsGFP-treated adults. In total, 20 treated adults were mixed and used for protein extraction. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control. (C) Immunofluorescence in midgut, salivary glands and ovaries of viruliferous planthopper treated with dsLstrE2 E and dsGFP. Anti-RSV CP (Alexa Fluor 488, green) and DAPI (blue) were used as fluorescent probes. Each treatment was replicated five times. Abbreviations: mg, midgut; sg, salivary glands; o, ovary; vm, visceral muscle; me, midgut epithelium; psg, principal salivary glands; gr, germarium. Bar = 50 µm.

LstrE2 E Does Not Directly Interact with RSV Proteins
Considering that LstrE2 E may mediate RSV load by binding viral proteins, we used yeast two-hybrid assays to evaluate whether LstrE2 E interacts with seven known RSV proteins (CP, SP, NS2, NS3, NSvc2, NSvc4, and RdRp). Full-length LstrE2 E was used as bait, and each of the six intact proteins (CP, SP, NS2, NS3, NSvc2, and NSvc4) and five truncated RdRp mutants were used as prey. All yeast strains failed to grow on synthetic dextrose dropout medium (Figure 8, Figure S2). This result suggested that LstrE2 E does not directly interact with RSV proteins.

Discussion
Phylogenetic analyses showed that 263 E2 enzymes from eight insect species could be categorized into 19 groups (Figure 1). There are few studies of insect E2s; consequently, the nomenclature and classification of E2s in insects is chaotic. Thus, we adopted nomenclature for small brown planthopper UBC-domain-containing proteins based on their relatedness to Drosophila and human orthologues [44]. LstrE2 E and LstrEff occur in higher eukaryotes as UBE2D1-4 and UBE2E1-3, which are involved in the degradation of short-lived and abnormally folded proteins [45]. The LstrE2 A/LstrE2 B and LstrE2 G1/LstrE2 G2 proteins also appear pairwise in humans [46]. LstrBruce, which encodes giant E2 protein, is present in both Drosophila and humans [47], whereas E2 O and E2 T have not been identified in small brown planthopper. Insect E2s in the same clade share a similar structure, which suggests that they may have related functions and possibly target similar lysine residues to construct polyubiquitin chains.
The UBC E2 family has expanded and diversified during evolution, and many E2s exist in both prokaryotes and eukaryotes. The E2 family includes both proteins and inactive variants that range in number from up to 20 in prokaryotes and over 40 in multicellular eukaryotes. For example, 12 and 16 E2s were identified in the algae Ostreococcus tauri and yeast Saccharomyces cerevisiae, respectively [44,48]; in plants, 37, 48, and 75 E2s were identified in Arabidopsis thaliana, rice, and maize, respectively [47][48][49]. Twenty and 37 E2s were identified in Caenorhabditis elegans and humans [11,46]. In this study, 20 E2s were initially identified in planthopper via transcriptome analysis; 18 were ubiquitinconjugating enzymes and two were Ubl-conjugating enzymes (SUMO-and NEDD8-conjugating enzymes) (Figure 1). The number of E2s in planthopper is smaller than the numbers in plants and humans, which is likely due to developmental complexity.
Expression profile analysis revealed that the four LstrE2s are present in all planthopper tissues and exhibit development-and tissue-specific expression patterns (Figures 3 and 4). The expression of the four LstrE2s peaked at three days after molting in planthopper adults, suggesting that they function in ubiquitination at this developmental stage. LstrE2 A/E/H were primarily expressed in planthopper midgut and were highly expressed during RSV infection; these results suggest a role for LstrE2 A/E/H in RSV infection in the midgut. Based on the high expression of LstrE2 G2 in ovaries and after RSV infection, we speculate that LstrE2 G2 may be involved in transovarial transmission of RSV. These results demonstrate that the four LstrE2s play important roles in the planthopper immune response, which warrants further investigation.
We show that LstrE2 E was highly expressed in response to RSV infection, and repression of LstrE2 E facilitated RSV accumulation in the planthopper body. These findings indicated that the role

Discussion
Phylogenetic analyses showed that 263 E2 enzymes from eight insect species could be categorized into 19 groups (Figure 1). There are few studies of insect E2s; consequently, the nomenclature and classification of E2s in insects is chaotic. Thus, we adopted nomenclature for small brown planthopper UBC-domain-containing proteins based on their relatedness to Drosophila and human orthologues [44]. LstrE2 E and LstrEff occur in higher eukaryotes as UBE2D1-4 and UBE2E1-3, which are involved in the degradation of short-lived and abnormally folded proteins [45]. The LstrE2 A/LstrE2 B and LstrE2 G1/LstrE2 G2 proteins also appear pairwise in humans [46]. LstrBruce, which encodes giant E2 protein, is present in both Drosophila and humans [47], whereas E2 O and E2 T have not been identified in small brown planthopper. Insect E2s in the same clade share a similar structure, which suggests that they may have related functions and possibly target similar lysine residues to construct polyubiquitin chains.
The UBC E2 family has expanded and diversified during evolution, and many E2s exist in both prokaryotes and eukaryotes. The E2 family includes both proteins and inactive variants that range in number from up to 20 in prokaryotes and over 40 in multicellular eukaryotes. For example, 12 and 16 E2s were identified in the algae Ostreococcus tauri and yeast Saccharomyces cerevisiae, respectively [44,48]; in plants, 37,48, and 75 E2s were identified in Arabidopsis thaliana, rice, and maize, respectively [47][48][49]. Twenty and 37 E2s were identified in Caenorhabditis elegans and humans [11,46]. In this study, 20 E2s were initially identified in planthopper via transcriptome analysis; 18 were ubiquitin-conjugating enzymes and two were Ubl-conjugating enzymes (SUMO-and NEDD8-conjugating enzymes) (Figure 1). The number of E2s in planthopper is smaller than the numbers in plants and humans, which is likely due to developmental complexity.
Expression profile analysis revealed that the four LstrE2s are present in all planthopper tissues and exhibit development-and tissue-specific expression patterns (Figures 3 and 4). The expression of the four LstrE2s peaked at three days after molting in planthopper adults, suggesting that they function in ubiquitination at this developmental stage. LstrE2 A/E/H were primarily expressed in planthopper midgut and were highly expressed during RSV infection; these results suggest a role for LstrE2 A/E/H in RSV infection in the midgut. Based on the high expression of LstrE2 G2 in ovaries and after RSV infection, we speculate that LstrE2 G2 may be involved in transovarial transmission of RSV. These results demonstrate that the four LstrE2s play important roles in the planthopper immune response, which warrants further investigation. We show that LstrE2 E was highly expressed in response to RSV infection, and repression of LstrE2 E facilitated RSV accumulation in the planthopper body. These findings indicated that the role of LstrE2 E in RSV accumulation is consistent with the role of animal and plant E2s in host defense mechanism against virus, as described above. However, Y2H analysis showed that LstrE2 E did not directly interact with RSV proteins. LstrE2 E inhibited RSV accumulation through an unknown antiviral defense mechanism. The human E2 enzyme UbcH7 functioned with the E3 SCF complex to ubiquitinate and degrade papillomavirus E7 protein [50], and the plant E2 Ubc3 and E3 Ligase RFP1 coordinately provide an antiviral mechanism that targeted a viral protein for degradation [17,18]. Based on these reports, it is plausible that LstrE2 E may also function with E3 to degrade viral proteins, thereby inhibiting viral accumulation. Another possibility is that LstrE2 E may impact viral load by an unknown pathway. The precise mechanism of RSV inhibition by LstrE2 E warrants further investigation.
In this study, 20 E2s were identified in small brown planthopper, and full-length cDNAs were obtained for four LstrE2s (LstrE2 A/E/G2/H). Expression of the four LstrE2s was highest in the midgut and ovaries of planthopper adults. LstrE2 E was highly expressed during RSV infection and inhibited viral accumulation in small brown planthopper. These results suggested that LstrE2 E expression correlated with RSV accumulation in small brown planthopper. These results provide insights for understanding the interaction between RSV and small brown planthopper and provide new avenues to control plant disease.

Conflicts of Interest:
The authors declare no conflict of interest.