Functional characterization of piggyBac-like elements from Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Functional characterization of piggyBac -like elements from Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) first-author https://orcid.org/0000-0002-5119-1658 1 Wenqing ZHANG Wenqing ZHANG State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China PiggyBac is a transposable DNA element originally discovered in the cabbage looper moth ( Trichoplusia ni ). The T. ni piggyBac transposon can introduce exogenous fragments into a genome, constructing a transgenic organism. Nevertheless, the comprehensive analysis of endogenous piggyBac -like elements ( PLE s) is important before using piggyBac , because they may influence the genetic stability of transgenic lines . Herein, we conducted a genome-wide analysis of PLE s in the brown planthopper (BPH) Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), and identified a total of 28 PLE sequences. All N. lugens piggyBac -like elements ( NlPLE s) were present as multiple copies in the genome of BPH. Among the identified NlPLE s, NlPLE25 had the highest copy number and it was distributed on five chromosomes. The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals, as well as a single open reading frame transposase encoding 546 amino acids. Furthermore, NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision. A cross-recognition between the NlPLE25 transposon and the piggyBac transposon was also revealed in this study. These findings provide useful information for the construction of transgenic insect lines. 目的 2 鉴定褐飞虱中的piggyBac类似元素并评估其转座活性。创新点　首次鉴定了褐飞虱中的piggyBac类似元素并发现NlPLE25具备转座活性。 方法 2 使用RepeatMasker和RepeatModeler软件预测褐飞虱基因组中的piggyBac类似元素序列。随后在S2细胞中测试了NlPLE25的切割活性，在Sf9细胞中测试了NlPLE25的转座能力。 结论 2 我们从褐飞虱基因组中鉴定出28个piggyBac类似元素。进一步分析NlPLE25发现其在昆虫细胞中引起精确的切除和转座。此外，我们还发现了NlPLE25与piggyBac转座子之间的交叉识别。 褐飞虱 piggyBac 类似元素 转座子 NlPlE25 Nilaparvata lugens piggyBac -like elements Transposon NlPLE25 1 Introduction 1 The piggyBac transposon, originally discovered in the cabbage looper moth ( Trichoplusia ni ), is 2472 bp in length and comprises three parts: a 13-bp terminal inverted repeat (TIR), a 19-bp asymmetric sub-terminal inverted repeat (STIR), and a 1782-bp transposase open reading frame (ORF) ( Cary et al., 1989 Cary et al., 1989 ; Fraser et al., 1996 Fraser et al., 1996 ). Its general features shared by various members of the piggyBac superfamily include the TTAA tandem site duplication (TSD), a triple-aspartate (DDD) motif, and a C-terminal cysteine-rich domain (CRD) in the piggyBac transposase core region ( Sarkar et al., 2003 Sarkar et al., 2003 ; Keith et al., 2008 Keith et al., 2008 ). Numerous transposons similar to piggyBac exist in a variety of organisms, known as piggyBac -like elements ( PLE s). Previous study revealed that the silkworm ( Bombyx mori ) has 98 PLE s, of which five PLE s contain a single ORF and TIRs ( Xu et al., 2006 Xu et al., 2006 ). Subsequently, yabusame , another PLE identified in the Bombyx mori genome, showed the almost complete loss of its transposition activity in vitro ( Daimon et al., 2010 Daimon et al., 2010 ). Moreover, nine PLE s were found in the cotton aphid ( Aphis gossypii ), of which AgoPLE1.1 has an intact transposase ORF ( Luo et al., 2011 Luo et al., 2011 ). The PLE-wu was identified in Sf9 cells, which exhibits transposition activity in insect and mammalian cells ( Wu and Wang, 2014 Wu and Wang, 2014 ). Certain PLE s were also reported in tobacco budworm ( Heliothis virescens ) ( Wang et al., 2006 Wang et al., 2006 ), cotton bollworm ( Helicoverpa armigera ) ( Sun et al., 2008 Sun et al., 2008 ), pink bollworm ( Pectinophora gossypiella ) ( Wang et al., 2010 Wang et al., 2010 ), and red flour beetle ( Tribolium castaneum ) ( Wang et al., 2008 Wang et al., 2008 ). PiggyBac transposons with high transpositional efficiency are commonly used in the germline transformation of many insects, such as the fruit fly ( Drosophila melanogaster ), silkworm ( B. mori ), yellow fever mosquito ( Aedes aegypti ), honeybee ( Apis mellifera ), pink bollworm ( P. gossypiella ), and Asian corn borer ( Ostrinia furnacalis ), with success rates of 0.1%–10% ( Gregory et al., 2016 Gregory et al., 2016 ). The brown planthopper (BPH) Nilaparvata lugens is one of the most destructive insect pests of rice ( Oryza sativa ) in Asian countries. A major hurdle to BPH research is the lack of molecular tools required for its genetic analysis. Although the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system ( Li et al., 2021 Li et al., 2021 ) has been used for gene knock-out in BPH ( Xue et al., 2018 Xue et al., 2018 ; Zhao et al., 2019 Zhao et al., 2019 ; Chen et al., 2021 Chen et al., 2021 ), knock-in has not yet been reported. PiggyBac is a potentially valuable alternative tool to solve this challenge; however, endogenous PLE s might affect the stability of the piggyBac transposition system integrated into the genome ( Jia et al., 2021 Jia et al., 2021 ). Therefore, a comprehensive analysis of endogenous PLE s is essential prior to the application of piggyBac. Previously, two versions (GCA_000757685.1 and GCA_014356525.1) of the whole BPH genome have been released. Here, we report a genome-wide analysis of PLE s in BPH. Special attention was paid to NlPLE25 , which has the highest number of copies in the genome. Our findings should contribute to the understanding of the distribution and characteristics of members of the piggyBac superfamily and their application in insects. 2 Materials and methods 1 2.1 Insect and cell cultures 2 A BPH population was reared on seedlings of the susceptible rice variety Huanghuazhan. The population was then maintained in a greenhouse at Sun Yat-sen University, Guangzhou, China, at (26±0.5) °C with (80±10)% relative humidity under a 16-h light/8-h dark photoperiod. Drosophila S2 cells were cultured in Schneider's Drosophila Medium (Thermo Fisher Scientific, Waltham, MA, USA) at 28 °C. Sf9 cells were cultured in Sf-900 II serum-free medium (Thermo Fisher Scientific) at 28 °C. 2.2 Identification of PLE s 2 The N. lugens assembly GCA_014356525.1 was adopted in this study. The RepeatModeler 5.8.8 ( http://www.repeatmasker.org http://www.repeatmasker.org ) and RepeatMasker 4.1.1 ( http://www.repeatmasker.org http://www.repeatmasker.org ) packages were employed to identify repeat families in BPH de novo. The interspersed repeat databases screened by RepeatMasker were based on the Repbase Update ( Bao et al., 2015 Bao et al., 2015 ) and Dfam ( Hubley et al., 2016 Hubley et al., 2016 ). To gain more access to sequences of PLE s from the BPH genome, a search was carried out of the BPH nucleotide database of the National Center for Biotechnology Information (NCBI) using the key phrase " piggyBac -like elements." The 3-kb sections upstream and downstream of each sequence were extracted to look for TIRs ( Bouallègue et al., 2017 Bouallègue et al., 2017 ). The sequence was considered a complete PLE when TIRs were found on both sides. The TIR sequence logo was created using WebLogo ( http://weblogo.berkeley.edu/logo.cgi http://weblogo.berkeley.edu/logo.cgi ). Multiple sequence alignment and phylogenetic tree construction were carried out using MEGA 6.0 ( https://www.megasoftware.net https://www.megasoftware.net ). TBtools ( Chen CJ et al., 2020 Chen CJ et al., 2020 ) was used to map the location of NlPLE s on chromosomes. PSORT II ( https://www.genscript.com/psort.html?src=leftbar https://www.genscript.com/psort.html?src=leftbar ) was utilized to predict the nuclear localization signal (NLS). 2.3 NlPLE25 amplification and analysis 2 The genomic DNA was isolated from BPH using an Omega insect DNA extraction kit (Omega Bio-Tek, Norcross, GA, USA) and quantified using a NanoDrop ND-2000 instrument (Thermo Fisher Scientific). The primers were designed using NCBI primer-Blast ( http://www.ncbi.nlm.nih.gov/tools/primer-blast http://www.ncbi.nlm.nih.gov/tools/primer-blast ) based on the NlPLE25 reference sequences in the NCBI database (Table S1). Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) was employed to amplify the DNA fragments used for cloning according to the manufacturer's instructions. The amplified fragments were cloned using pEASY-Blunt Zero (TransGen Biotech, Beijing, China). Sequencing was performed by Tianyi Huiyuan Biotech (Wuhan, China), and the data were analyzed using DNAMAN 8.0 (LynnonBiosoft, Quebec, Canada). The obtained pEASY- NlPLE25 vector was used in this study. 2.4 Plasmid construction 2 Three plasmids (pEGFP, p NlPLE25 -EGFP, and pReplace-EGFP) were constructed for the excision assays. The pIEX/Bac1 vector was purchased from Youbio Biotech (Changsha, China). The enhanced green fluorescent protein (EGFP) was placed under the regulation of homologous region 5 (hr5) enhancer, IE1 promoter, and P10 promoter in pIEX/Bac1 by homologous recombination cloning to construct the pEGFP vector. The homologous recombination-based cloning method was performed using the In-Fusion HD Cloning Kit (TaKaRa Bio, Kusatsu, Japan). Similarly, full-length NlPLE25 was inserted between the promoter and the EGFP to construct the p NlPLE25 -EGFP, whereas the N. lugens carboxylesterase precursor gene (MF278673.1, 1.64kb) was cloned into p NlPLE25 -EGFP, replacing the NlPLE25 transposase ORF, in order to construct pReplace-EGFP. For the transposition assays, four reporter plasmids (pPB-BleoR, p NlPLE25 -BleoR, p NlPLE25 , and pPB) were constructed. For pPB-BleoR and p NlPLE25 -BleoR, the OpIE2-driven bleomycin resistance gene from PIZ/V5-His was ligated into the flanking sequences of the TIRs and STIRs of piggyBac and NlPLE25 transposons, respectively, to build transposon reporters. The flanking sequences were obtained from pXL-BacII-3XP3-RFP and pEASY- NlPLE25 , respectively. To construct the transposase expression vectors p NlPLE25 and pPB, the NlPLE25 transposase and T. ni piggyBac transposase were cloned into pIEX/Bac1 vectors. All cloning experiments were performed using the In-Fusion HD Cloning Kit (TaKaRa Bio). The primers for vector construction are listed in Table S1. 2.5 Excision assays 2 The excision activity was tested using S2 cells . EGFP cannot be expressed in p NlPLE25 -EGFP unless the NlPLE25 transposase protein removes the full-length of NlPLE25 from the sequence end TTAA. pReplace-EGFP was used as negative control and pEGFP as positive control. Prior to transfection, the S2 cells were seeded into 6-well plates and cultured at 28 °C overnight. At 80%–90% confluence, cells were transfected using FuGENE HD (Promega, Madison, WI, USA) with 2 µg of plasmid. The green fluorescence was visualized under a fluorescent microscope (Leica Biosystems, Nussloch, Germany) at 48 h post-transfection, and green fluorescence cells and total cells were counted in 10 fields at 200× magnification. The ratio of green fluorescence cell number to total cell number reflects the excision efficiency of NlPLE25 . The DNA was extracted using the Omega Tissue DNA Extraction Kit (Omega Bio-Tek) at 48 h post-transfection for sequence analysis. Primer pairs were designed based on adjacent areas of NlPLE25 on p NlPLE25 -EGFP (Table S1), and polymerase chain reaction (PCR) amplification was carried out as described in Section 2.3. 2.6 Resistance colony formation assay 2 Sf9 cells were divided into six groups for transfection. The two control groups were pPB-BleoR and p NlPLE25 -BleoR, and the four experimental groups were: (1) p NlPLE25 and p NlPLE25 -BleoR; (2) pPB and pPB-BleoR; (3) p NlPLE25 and pPB-BleoR; and (4) pPB and p NlPLE25 -BleoR. The transfection and excision assays were carried out as described in Section 2.5. Upon transfection, cells were seeded at a density of 1×10 6 cells per 10-cm dish and selected with 0.2% (2 g/L) Zeocin (Jingxin Biotech, Guangzhou, China) for two weeks. The resultant colonies were fixed with 10% (volume fraction) methyl alcohol and stained with 0.1% (1 g/L) crystal violet (Macklin Biochemical, Shanghai, China). To detect the presence of plasmids in cells after drug screening, 500 ng of purified DNA was used to transform Escherichia coli DH5α competent cells. The Luria-Bertani plates were then incubated at 37 °C overnight. 2.7 RT-PCR 2 After two weeks of selection, the messenger RNA (mRNA) levels of the bleomycin resistance gene were measured using reverse transcription (RT)-PCR. The RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific), according to the manufacturer's instructions, and used for reverse transcription by the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) kits. PCR amplification was carried out as described in Section 2.3. The primers for RT-PCR are listed in Table S1. 2.8 Inverse PCR 2 The genomic DNA from Sf9 cells was digested with Hae III. The digestion products were ligated, generating circular molecules containing either the right end or the left end of the transposon. PCR amplification was carried out as described in Section 2.3. The primers for inverse PCR are listed in Table S1. 2.9 Spatial and temporal expression patterns of NlPLE25 2 The total RNA from the seven developmental stages (egg, 1st instar nymph, 2nd instar nymph, 3rd instar nymph, 4th instar nymph, 5th instar nymph, and adult female) and six adult tissues (head, fat body, cuticle, leg, midgut, and ovary) of insects was extracted and reverse transcribed as described in Section 2.7. Quantitative real-time PCR (qPCR) was carried out using the Roche 480 Real-Time PCR System (Roche, Basile, Switzerland) with SYBR Green qPCR Master Mix (EZBioscience, Roseville, MN, USA). The primers for real-time quantitative PCR are listed in Table S1. 2.10 Data analysis 2 Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software; https://www.graphpad.com https://www.graphpad.com ). For the excision activity of NlPLE25 , statistical significance was assessed with paired t -test. A value was considered statistically significant at P < 0.05, and highly significant at P < 0.01. For the frequency of NlPLE25 transposase integration, the average values from all the independent replicates are shown in the figures. Data are represented as mean±standard error ( n =5). Different lower-case letters above the columns represent significant difference at P < 0.05. 3 Results 1 3.1 PiggyBac superfamily in BPH 2 The results revealed that the BPH genome assembly was composed of 59.06% of repeats, including 10.26% retroelements, 2.90% DNA transposons, 1.90% simple repeats, 0.35% low-complexity regions, and 43.65% unclassified regions ( Table 1 Table 1 ). The 988 piggyBac sequences predicted from DNA transposons had a total length of 316 836 bp, which accounts for 0.03% of the BPH genome ( Table 1 Table 1 ). The TBLASTn analysis revealed that 55 homologs of T. ni piggyBac were present in the BPH genome. Subsequently, we performed TTAA and TIR searches on the 3-kb upstream and downstream positions of these sequences, and filtered out sequences with ORFs coding for amino acid residues of < 250. When TIRs were found on both ends of an ORF, the sequence was considered to be a PLE , while the remaining sequence was considered to be a piggyBac -derived ( PGBD ) element or PGBD -like element. Finally, 28 sequences with perfect flanks were determined and named as NlPLE1 – NlPLE28 ( Table 2 Table 2 ). SINE s: short interspersed elements; LINE s: long interspersed elements; SLACS : spliced leader associated conserved sequence; L1 : LINE s 1; L2 : LINE s 2; CR1 : chicken repeat 1; Rex: retroelement of Xiphophurus ; LTR : long terminal repeat. Class Number Length (bp) Ratio of genome (%) DNA transposons 64 016 31 563 456 2.90 Hobo-Activator 4818 1 544 728 0.14 Tc1-IS630-Pogo 28 786 10 196 413 0.94 En-Spm 0 0 0.00 MuDR-IS905 0 0 0.00 PiggyBac 988 316 836 0.03 Tourist / Harbinger 5626 1 516 002 0.14 Other (Mirage , P-element , Transib) 988 568 632 0.05 Retroelements 219 959 111 649 813 10.26 SINEs 0 0 0.00 Penelope 18 531 5 300 981 0.49 LINEs 191 954 80 571 250 7.41 CRE / SLACS 0 0 0.00 L2 / CR1 / Rex 124 335 53 275 704 4.90 R1 / LOA / Jockey 403 211 231 0.02 R2 / R4 / NeSL 1457 702 984 0.06 RTE / Bov-B 32372 13 096 311 1.20 L1 / CIN4 0 0 0.00 LTR 28 005 31 078 563 2.86 BEL / Pao 8446 11 603 870 1.07 Ty1 / Copia 1351 648 582 0.06 Gypsy / DIRS1 18 208 18 826 111 1.73 Retroviral 0 0 0.00 Rolling circles 0 0 0.00 Unclassified 1 941 732 474 795 500 43.65 Total interspersed repeats 618 008 769 56.81 * Percent (%) indicates sequence similarity to Trichoplusia ni piggyBac transposase. ORF: open reading frame. Name Full length (bp) ORF length (bp) Amino acid length 5' TTAA TIR Percentage identity (%) * Copies NlPLE1 3383 1101 367 TTAACCCTTAATTACTAGGGATG 6.23 1 NlPLE2 2942 1953 651 TTAACCCTCTAGTGCAT 15.92 6 NlPLE3 3259 1173 391 TTAACCCTTTATTGCATG 15.33 2 NlPLE4 2527 921 307 TTAACACTCCGCTGCTCGC 26.69 3 NlPLE5 2848 2022 674 TTAACCCTTAATTACTACAA 15.17 3 NlPLE6 2921 1887 629 TTAACCCTACAATGCAT 9.63 2 NlPLE7 2808 1983 661 TTAACCCTACAATGCATAAT 8.86 2 NlPLE8 2990 1413 471 TTAACCCTTTAATGCATG 15.07 4 NlPLE9 3328 1209 403 TTAACCCTTCAAGTGATGGGTG 14.33 2 NlPLE10 2284 759 253 TTAACCCTTAGCGCTCG 11.00 1 NlPLE11 3173 1257 419 TTAACCCTACACCGCATG 7.30 2 NlPLE12 2967 1950 650 TTAACCCTCTAGTGCAT 12.71 6 NlPLE13 3213 1809 603 TTAACCCTTTATTGCACAAT 14.05 4 NlPLE14 2953 1887 629 TTAACCCTCGGGCACAGGCAC 8.58 2 NlPLE15 3241 771 257 TTAACCCTTAGTTACTATC 18.59 1 NlPLE16 3001 804 268 TTAACCCTTAAATGCATG 18.44 4 NlPLE17 2951 1014 338 TTAACACTATTACCGAAACCC 20.07 5 NlPLE18 2401 1146 382 TTAACCCTGCATTACTCGCGCGGG 23.60 4 NlPLE19 2762 1875 625 TTAACCCTTTGCGATC 18.22 2 NlPLE20 3337 1008 335 TTAACCCTCAATTACTACGGA 19.48 1 NlPLE21 3092 993 330 TTAACACGTGAGTACACGCCCCC 17.31 3 NlPLE22 2412 1887 628 TTAACCCTTTAATGCATG 12.20 4 NlPLE23 3247 1899 632 TTAACCCTTAGTTACTATC 27.46 1 NlPLE24 5215 1704 567 TTAACCCTGCGGTACTCGCGCGGGCTA 32.28 5 NlPLE25 2590 1761 586 TTAACCCTCCGGTAGGCGCGCGG 30.25 8 NlPLE26 2656 930 309 TTAACCCTTTATAAGGTAATGGCAGA 10.45 1 NlPLE27 2088 1563 520 TTAACCCAGTAAGAACCACGT 28.67 4 NlPLE28 2424 1899 632 TTAACCCTTAATTACTACAAGC 27.50 3 Each of the 28 NlPLE s had a total length of 2088–5215 bp with an ORF of 759–2022 bp ( Table 2 Table 2 ). The length of TIRs ranged between 15 and 23 bp, which is slightly longer than that of the 12–19 bp previously reported by Fraser et al. (1983) Fraser et al . (1983) . The first six nucleotides were relatively well conserved following the motif C[ C /A]C[ T /A/G][ T /C/A][ T /A/C] ( Fig. 1 Fig. 1 a). Each NlPLE had one or more copies in the genome ( Table 2 Table 2 and Fig. 1 Fig. 1 b) distributed across the BPH chromosomes, except for NlPLE14 , which exhibited the highest enrichment in chromosome 1 ( Fig. 1 Fig. 1 b). The amino acid sequence alignment showed that transposases of the 28 NlPLE s had a similarity of 6.23%–32.28% with those of the T. ni piggyBac ( Table 2 Table 2 and Fig. 1 Fig. 1 c). NlPLE24 and NlPLE1 showed the highest (32.28%) and lowest (6.23%) sequence similarity, respectively. The similarity of NlPLE amino acid sequences ranged between 1.98% and 62.12% (Table S2). To confirm the accurate phylogenetic relationships of NlPLE transposases, 157 previously described PLE transposases were selected from different genomes for the construction of the phylogenetic tree. Despite the divergence, the sequence analysis showed that most PLE transposases were found to contain conserved domains for a total of about 300 residues that were roughly delimited by the putative DDD motif and conserved cysteine residues ( Figs. 1 Figs. 1 c and S1). Thus, a phylogenetic tree was constructed based on these conserved regions using MEGA. The simplified phylogeny is shown in Fig. 1 Fig. 1 d (see Fig. S2 for the complete tree). The NlPLE transposases were classified into six major groups. The largest clade included 14 NlPLE s and was clustered together with the T. ni piggyBac . Furthermore, we found that NlPLE17 and NlPLE25 showed a close evolutionary relationship with T. ni piggyBac. The NlPLE s identified via whole-genome BLAST analysis were widely distributed throughout the BPH genome. NlPLE25 was one of the most prominent NlPLE s, sharing a 30.25% similarity with T. ni piggyBac transposase. Therefore, it was selected for further study. 3.2 Structure and distribution of NlPLE25 2 The NlPLE25 was found to be 2582-bp long and flanked by typical tetranucleotide target site TTAA duplications ( Fig. 2 Fig. 2 a). Both ends of NlPLE25 contain a 16-bp TIR and a 20-bp STIR, which are asymmetric with a 19-bp spacer at the 3' end and a sequence duplication between segments relative to the 5' end ( Fig. 2 Fig. 2 a). The TIRs and STIRs of NlPLE25 had a 50% similarity with T. ni piggyBac ( Fig. 2 Fig. 2 b). The NlPLE25 had an ORF encoding a 586-amino acid transposase ( Fig. 2 Fig. 2 c), and a bipartite NLS (RRCQIDGLPVQLKKRGK) with a score of 0.89 detected using PSORT II. The NlPLE25 transposase contains one N-terminal domain (NTD), one CRD, two dimerization and DNA-binding domains (DDBDs), and one catalytic domain with an insertion domain (Figs. 2b 2b and 2c 2c ). The catalytic domain contains a conserved DDD motif (D263, D343, and D446), consistent with the amino acids of D268, D346, and D447 in T. ni piggyBac transposase ( Fig. 2 Fig. 2 c). The chromosomal location and copy number of NlPLE25 were determined using the NCBI databank ( Fig. 1 Fig. 1 b and Table 2 Table 2 ). The BLAST results showed that NlPLE25 exists with at least eight copies in the BPH genome, distributed on five chromosomes (1, 3, 7, 10, and X). These copies were designated as NlPLE25.1 – NlPLE25.8 ( Fig. 2 Fig. 2 e) . Five out of eight copies had the same sequence. Two copies ( NlPLE25.6 and NlPLE25.8 ) exhibited insertions and/or deletions (indels); the indels of NlPLE25.6 suggested a premature translation termination, and the coding region of NlPLE25.8 was disrupted because of the indels. The flanking sequences of NlPLE25 , except for NlPLE25.1 and NlPLE25.8 , were localized at sites with a consensus TTAA, which is characteristic of piggyBac family members. The expression of NlPLE25 transposase was significantly higher in the 5th instar nymph than in other stages, whereas the lowest expression was detected in the 3rd and 4th instars (Fig. S1a). NlPLE25 was mainly expressed in the fat body, with significantly higher levels than those in other tissues (Fig. S1b). 3.3 Excision activity of NlPLE25 in S2 cells 2 Since NlPLE25 and T. ni piggyBac were found structurally similar ( Fig. 2 Fig. 2 c), it was hypothesized that NlPLE25 might have functions analogous to T. ni piggyBac . In p NlPLE25 -EGFP, the expression of EGFP was blocked by the full-length NlPLE25 until its removal by the expressed transposase ( Fig. 3 Fig. 3 a). Thus, no green fluorescence was observed when pReplace-GFP was transfected ( Fig. 3 Fig. 3 b). However, 1.26% and 40.61% of the cells exhibited green fluorescence when p NlPLE25 -EGFP and pEGFP were transfected, respectively (Figs. 3b 3b and 3c 3c ). In addition, DNA amplification produced an approximately 500-bp band, and sequencing revealed the traceless excision of NlPLE25 (Figs. 3d 3d and 3e 3e ). 3.4 DNA transposition activity of NlPLE25 2 It was assumed that NlPLE25 would retain its transposase activity because of its traceless excision activity. This was examined by analyzing NlPLE25 transposition in cultured Sf9 cells using a synthetic transposon reporter in a transposase expression plasmid co-transfection assay. The synthetic transposon reporter was comprised of a bleomycin resistance gene flanked by TIRs and STIRs of the transposon ( Fig. 4 Fig. 4 a). Cells were subjected to the excision assay two days after transfection. Six groups were included in the assay: two groups for single transfection (L1 and L2; Fig. 4 Fig. 4 b), and four groups for co-transfection (L3–L6; Fig. 4 Fig. 4 b). All of the co-transfected groups (L3–L6) were successfully excised, whereas such changes were not observed in the single transfection groups (L1 and L2; Fig. 4 Fig. 4 b). In L4 and L6, NlPLE25 and T. ni piggyBac transposases recognized the TIRs and STIRs of each other ( Fig. 4 Fig. 4 b). The clonogenic assay revealed significant rates of bleomycin resistance of cells conferred by the transposon reporter with transposase expression plasmids; however, bleomycin resistance was not observed in cells expressing a control lacking the transposase (Figs. 4c 4c and 4d 4d ). The relative integration frequency in the pPB+pPB-BleoR group was significantly higher than that in the other groups. In contrast, no significant differences were observed between any two of the three groups (pPB+p NlPLE25 -BleoR, p NlPLE25 +p NlPLE25 -BleoR, and p NlPLE25 +pPB-BleoR) ( Fig. 4 Fig. 4 d). The frequency of NlPLE25 transposase integration was 30.5% of that of T. ni piggyBac ( Fig. 4 Fig. 4 d). The RT-PCR results confirmed the stable expression of the bleomycin resistance gene in the four co-transfection groups ( Fig. 4 Fig. 4 e). Inverse PCR performed on the four lines generated using the four different co-transfection groups revealed that the 5' insertion sequence matched the 3' sequence ( Fig. 4 Fig. 4 f), which argued for single insertions. Thus, the observed results indicated that NlPLE25 retained transposon activity and could act on the TIRs and STIRs of T. ni piggyBac , probably due to the high sequence similarity in the TIRs and STIRs between NlPLE25 and T. ni piggyBac ( Fig. 2 Fig. 2 b). 1,2;3;4;5;6; 4 Discussion 1 In the present study, 28 NlPLE s with perfect TSDs were identified in the BPH genome using different methods and bioinformatics tools. These NlPLE s retained all the canonical features of the piggyBac superfamily, including TIRs at the extreme ends, and an ORF encoding a transposase. An NlPLE was observed to always insert into TTAA target sites, similar to the results reported by Bouallègue et al. (2017) Bouallègue et al . (2017) . The sequence of the TIRs varies among different NlPLE s, but there was a notable conservation of the first six nucleotides C[ C /A]C[ T /A/G][ T /C/A][ T /A/C], which is also similar to the result of Bouallègue et al. (2017) Bouallègue et al . (2017) . Furthermore, the first three nucleotides in TIRs play an essential role in transposase recognition, since they are cleaved from the target site, and even a single nucleotide deletion is sufficient to abolish excision ( Elick et al., 1997 Elick et al., 1997 ). The sequence of the first three nucleotides is commonly CCC; however, other sequence types can also be recognized and cleaved by PLE transposase ( Elick et al., 1997 Elick et al., 1997 ). A more in-depth analysis of NlPLE25 allowed the identification of eight NlPLE25 copies, some of which had defects (Figs. 1b 1b and 2e 2e ). Incomplete fragments could be rapidly lost in the absence of trans -mobilization (le Rouzic et al., 2007; Hua-Van et al., 2011 Hua-Van et al., 2011 ) or domesticated into a functional protein ( Luo et al., 2014 Luo et al., 2014 ). Similar to T. ni piggyBac , NlPLE25 was comprised of two sections ( Fig. 2 Fig. 2 a): (1) asymmetric TIRs and STIRs at both ends, necessary for transposase to form the active synaptic complex in vivo ( Chen QJ et al., 2020 Chen QJ et al., 2020 ); and (2) a transposase ORF. The NlPLE25 transposase has similar functional domains to the T. ni piggyBac , including NTD, CRD, DDBD, and the catalytic domain (Figs. 2c 2c and 2d 2d ). The CRD is required for transposase activity ( Chen QJ et al., 2020 Chen QJ et al., 2020 ) and is frequently found in other PLE s. The catalytic domain of NlPLE25 transposase contains DDD motif, corresponding to D268, D346, and D447 in T. ni piggyBac transposase (Figs. 2c 2c and 2d 2d ). Furthermore, a putative NLS enables NlPLE25 transposase to enter the nucleus for DNA translocation catalysis ( Fig. 2 Fig. 2 c). Nevertheless, additional studies are required to validate the existence of this putative NLS. Our experiments confirmed the speculation that NlPLE25 might retain its transposase activity. The frequency of NlPLE25 transposase integration was 30.5% compared to that of T. ni piggyBac ( Fig. 4 Fig. 4 d). NlPLE25 transposase recognized the TIRs and STIRs of T. ni piggyBac , probably because of the similar transposon and transposase structures between NlPLE25 and T. ni piggyBac . A similar phenomenon was observed in human PGBD5 , the most evolutionarily conserved transposable element-derived gene in vertebrates, which can recognize T. ni piggyBac TIRs and catalyze DNA transposition in human cells ( Henssen et al., 2015 Henssen et al., 2015 ). Nevertheless, the transposase activity of NlPlE25 is a double-edged sword. On the one hand, it can be used as a potential insect transgenic tool. Hyperactive piggyBac transposase has seven amino acid substitutions related to T. ni piggyBac and is commonly used in transgenic applications, but its efficiency is not always high. On the other hand, NlPLE25 may influence the genetic stability of piggyBac transgenic lines. Endogenous PLE s may repress the transposition of introduced piggyBac vectors and their stability ( Lorenzen et al., 2003 Lorenzen et al., 2003 ; Jia et al., 2021 Jia et al., 2021 ). In the present study, NlPLE25 was found to share TIRs and STIRs with T. ni piggyBac ; therefore, NlPLE25 might transfer or remove foreign fragments in the piggyBac transgenic lines of BPH. The expression of NlPLE25 was observed in nearly all BPH tissues and throughout the BPH development period (Figs. S3a and S3b). If NlPLE25 removes foreign fragments of piggyBac from the genome of gonadal germline cells, the genetic stability of the transgenic lines will be destroyed. It is important to note that, beyond NlPLE s, other sequences flanked by a single TIR or no TIR exist, but these encode functional transposase in the BPH genome, and thus could still be capable of cross-reacting with integrated piggyBac transposons. Although host genomes have evolved multiple mechanisms to negatively regulate transposable element activity ( Reik, 2007 Reik, 2007 ; Slotkin and Martienssen, 2007 Slotkin and Martienssen, 2007 ; Jacobs et al., 2014 Jacobs et al., 2014 ; Sanchez-Luque et al., 2019 Sanchez-Luque et al., 2019 ), this risk should not be ignored. One of the potential limitations of this work is that it is necessary to assess the effect of endogenous PLE s on the stability of piggyBac transgenic lines in an in vivo system, as previously discussed. Future work might need to assess this risk by using piggyBac transgenic BPH lines. 5 Conclusions 1 This study aimed to identify endogenous PLE s in the BPH . We identified 28 PLE s and found that NlPLE25 had the highest copy number and was distributed across five chromosomes. NlPLE25 transposase achieved precise excision and transposition in cultured insect cells. We also indicated cross-recognition between the NlPLE25 transposon and the piggyBac transposon. The findings of this study will contribute to our understanding of the distribution and characteristics of members of the piggyBac superfamily and their application in insects. AcknowledgmenAcknowledgement:ts This work was supported by the Foundation of Guangzhou Science and Technology Key Project (No. 201904020041), China. Author contributioauthor contribution statement: ns Jun LV designed and performed the experiment, wrote and edited the manuscript. Qin SU, Jinhui LIU, Lin CHEN, and Jiawei SUN participated in part of the experiment and edited the manuscript. Wenqing ZHANG designed and supervised the experiment, wrote and edited the manuscript. All authors have read and approved the final version of the manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. References Bao WD , Kojima KK , Kohany O , 2015 . Repbase Update, a database of repetitive elements in eukaryotic genomes . 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Insect Biochem Mol Biol , 115 : 103246 . https://doi.org/10.1016/j.ibmb.2019.103246 https://doi.org/10.1016/j.ibmb.2019.103246 Mutations in NlInR1 affect normal growth and lifespan in the brown planthopper Nilaparvata lugens 2021-12-26 2022-03-03 2022-05-30 2022-04-15 © Zhejiang University Press 2022 Jun LV, Qin SU, Jinhui LIU, Lin CHEN, Jiawei SUN, and Wenqing ZHANG declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术）