The brown planthopper (BPH) Nilaparvata lugens is one of the most destructive insect pests in the rice fields of Asia. Like other hemipteran insects, BPH is not susceptible to Cry toxins of Bacillus thuringiensis (Bt) or transgenic rice carrying Bt cry genes. Lack of Cry receptors in the midgut is one of the main reasons that BPH is not susceptible to the Cry toxins. The main Cry-binding proteins (CBPs) of the susceptible insects are cadherin, aminopeptidase N (APN), and alkaline phosphatase (ALP). In this study, we analyzed and validated de novo assembled transcripts from transcriptome sequencing data of BPH to identify and characterize homologs of cadherin, APN, and ALP. We then compared the cadherin-, APN-, and ALP-like proteins of BPH to previously reported CBPs to identify their homologs in BPH. The sequence analysis revealed that at least one cadherin, one APN, and two ALPs of BPH contained homologous functional domains identified from the Cry-binding cadherin, APN, and ALP, respectively. Quantitative real-time polymerase chain reaction used to verify the expression level of each putative Cry receptor homolog in the BPH midgut indicated that the CBPs homologous APN and ALP were expressed at high or medium-high levels while the cadherin was expressed at a low level. These results suggest that homologs of CBPs exist in the midgut of BPH. However, differences in key motifs of CBPs, which are functional in interacting with Cry toxins, may be responsible for insusceptibility of BPH to Cry toxins. Key words: de novo assembly, brown planthopper, cadherin, aminopeptidase N, alkaline phosphatase The brown planthopper (BPH) Nilaparvata lugens is a widely distrib- Lack of proper receptors in the midgut of target insects has been uted insect pest in the rice fields of Asia. BPH can cause severe damage known to be a key reason for the insusceptibility of insect to Cry by feeding on phloem tissues and by transmitting viral diseases (Hibino toxins. Cry toxins are known to bind to cadherin-like proteins, gly- 1996, Jia et al. 2012). No effective environment-friendly control meth- cosylphosphatidylinositol (GPI)-anchored aminopeptidase N (APN), ods are currently available for BPH. Hence, the use of chemical pesticides GPI-anchored alkaline phosphatase (ALP), and ATP-binding cas- is the major means of management of BPH infestations (Lu et al. 2015). sette transporters after activation of protoxin in the process of their Bacillus thuringiensis (Bt) Cry toxins are well-known biological insecticidal activity (Pardo-López et al. 2013, Bravo et al. 2013). The agents for control of insect pests (Gómez et al. 2007, Vandenborre known Bt receptor proteins play critical roles in insect metabolism et al. 2011). Cry toxins have high insecticidal activity against lepidop- and are essential proteins of all insects. Hence, homologous proteins terans, coleopterans, and mosquitoes (Bravo et al. 2011). However, of Cry-binding protein (CBP) are likely to be present in the insects Cry toxins show no or very weak insecticidal activity on hemipteran which are not susceptible to Bt toxins. insects, such as planthopper, leafhopper, and aphids (Chougule and RNA-deep sequencing technology has become a powerful Bonning 2012, Shao et al. 2013). means for transcript discovery and measurements of transcriptional © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/10/4839024 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. XX, No. X expression levels. RNA sequencing has also been used in the iden- Kit for Illumina (NEB, USA) following the manufacturer’s recom- tification of receptor homologous genes of Bt toxins. For example, mendations. Fragment sizes of the prepared cDNA library were Liu et al. (2012) identified five APNs and four ALPs from the mid- selected by agarose gel electrophoresis and then were quantitated gut of Aphis glycines (Hemiptera: Aphididae) (Liu et al. 2012). Shu through qPCR by the use of Library Quantification Kit-Illumina GA et al. (2015) identified an APN from the Holotrichia parallela mid- Universal (Kapa, KK4824). The qualified cDNA libraries were clus- gut as a receptor of Cr y8Ea toxin (Shu et al. 2015). Transcriptome tered through the Illumina cbot system and sequenced on an Illumina sequencing of BPH has also been conducted and resulted in the iden- HiSeq 2500 platform to generate 125 nt paired-end reads (Biomarker tification of digestion, detoxification, and immune response-related Technologies Co., Ltd., Beijing, China). The original image data were genes from BPH guts (Peng et al. 2011, Bao et al. 2012). However, processed with Illumina GA Pipeline v1.3 to clean reads, followed homologs of potential Cry toxin receptor-related genes, e.g., cad- by the removal of adapter sequences, empty reads, and low-qual- herins, APNs, and ALPs, in the midgut of BPH have not yet been ity reads. A short reads assembling program from Trinity (version investigated. We have previously reported that Cry1Ab protoxin 2.1.1) was used for sequence assembly (Grabherr et al. 2011). To could be proteolytically processed by gut proteases of BPH but still evaluate the completeness of the assembly result, assembled contigs be nontoxic to BPH nymphs (Shao et al. 2013). Consequently, lack were analyzed by the BUSCO (Benchmarking Universal Single-Copy of proper receptor proteins in the midgut may be one reason for the Orthologs) program, based on the arthropod gene sets. low toxicity of Cry toxins to BPH. In this research, we investigated transcriptome-sequencing data of BPH to identify putative homolo- Sequencing Data Analysis gous genes of CBPs, followed by sequence verification by real-time The OrfPredictor program (Min et al. 2005) was used to obtain the polymerase chain reaction (RT-PCR) and expression-level calcula- open reading frame (ORF) of each unigene. Three ORF predictions tion by RT-qPCR. We also compared characteristics and structure were made for nucleotide sequence of each unigene, starting from of CBPs with their homologs in BPH. Our results indicated that the first, the second, and the third nucleotide with termination at homolog of CBPs exists in the midgut of BPH showing similar char- a stop codon, respectively. The ORF with the maximum size was acteristics and structure. selected as the final output for each sequence. Cleaned reads were mapped to unigenes by the Bowtie software (Langmead 2010), and then the number of reads mapping to each unigene as RPKM (Reads Materials and Methods Per Kilobase per Million mapped reads) was calculated through Insects the RSEM software (Li and Dewey 2011). Annotation of each The BPH strain used for this work was originally collected from unigene was conducted by searching all unigenes in the National rice fields of Fuzhou, Fujian, and has been reared in the laboratory Center of Biotechnology Information (NCBI) nr (nonredundant) conditions by the Institute of Plant Virology, Fujian Agriculture and database through the BLAST software (Altschul et al. 1997) with −5 Forestry University, Fuzhou, Fujian, China for more than 5 yr. The an E-value cutoff of 10 . Functional annotation by gene ontology BPH colony was maintained on rice (Oryza sativa L.) seedlings and (GO) was conducted by searching unigenes against the GO data- kept in a growth chamber at 28°C with a photoperiod of 14:10 base (Ashburner et al. 2000) through the Blast2GO software (http:// (light:dark) h. www.blast2go.org/). Sequences of putative cadherins, APNs, and ALPs have been deposited in the GenBank (the accession number of each gene is shown in Supp Table 1). Tissue Collection, RNA Isolation, and Illumina Sequencing Quantitative Real-Time PCR (RT-qPCR) Analysis and Over 200 third- to fourth-instar BPH nymphs were anesthetized Reverse PCR (RT-PCR) on ice for 10 min and dissected under an SMZ-B4 microscope (Chongqing Optec Instrument, China) to isolate the digestive tract. RT-qPCR was performed using the total RNA isolated from either The anterior diverticulum, hindgut, malpighian tubules, and esopha- the midgut or body of BPH, and assays were repeated with three gus were then removed before rinsing the midgut tissue with ice-cold biological replications. Total RNA of each replication from either diethyl pyrocarbonate (DEPC)-treated phosphate-buffered saline the midgut or body, extracted as described previously, was used (PBS) solution. The obtained midguts were stored in DEPC-treated for first-strand cDNA synthesis using a random primer and AMV PBS solution and kept in a −80°C freezer for further use. Midgut reverse transcriptase (TaKaRa, Japan). Efficiencies of each primer collection was repeated for three times. The midgut tissues were pair were verified to be around 96–110% before RT-qPCR assay. homogenized in a Dounce tissue grinder (Wheaton), and total RNA RT-qPCRs were carried out on an ABI 7500 real-time PCR detec- was extracted using an HP Total RNA Kit (Omega, USA) according tion system using 100 ng of the cDNA template or the nontem- to the manufacturer’s protocol. The quantity of extracted RNA was plate control (NTC), 0.2 μM of primers (Supp Table 2), and SYBR measured with a NanoDrop (Bio-Rad, USA), and the quality of RNA Premix Ex Taq (TaKaRa, Japan). PCR was performed with an initial was verified by an Agilent 2100 (Agilent, Germany) and electrophor - denaturation step at 95°C for 30 s, followed by 40 cycles at 95°C for esis gel analysis. Total RNA of the BPH body was prepared from 50 5 s, 55°C for 30 s, and a dissociation step added at the end. A melt- of the third- to fourth-instar nymphs by the previously described ing-curve analysis was performed upon completion of the real-time procedures with three replications. Small aliquots of RNA extracted PCR runs for each sample to examine the specificity of the PCR from samples were stored at −80°C for further analysis by RT-qPCR reactions. The melting curves from all sample running in this study assays. The rest of the RNA samples of either midgut or body were showed a single peak. After expression evaluation by the RefFinder pooled together, and 30 μg of each pooled sample was used for RNA online software (http://fulxie.0fees.us) (Xie et al. 2012), the BPH library preparation. 18s rRNA gene (GenBank Accession No. JN662398) was selected Purification of mRNA was performed using NEBNext Poly as an internal control by using the primers previously reported by (A) mRNA Magnetic Isolation Module (NEB, E7490). Sequencing Bao et al. (2012). RT-qPCR was repeated three times, and the results libraries were generated using NEBNext Ultra RNA Library Prep were standardized to the expression level of the BPH 18s rRNA Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/10/4839024 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. XX, No. X 3 gene. An NTC sample was run to detect any contamination and to assembled by Trinity assembler with the default parameter setting, determine the degree of dimer formation. Statistical analysis of the resulting in an assembly of 91,536 transcripts (≥200 nt) with a mean data was performed using one-way analysis of variance running by length of 1,177 bp. From the transcripts, 53,502 unigenes were SPSS software (version 22.0.0), along with online statistical tools predicted with a mean size of 856 bp (Supp Table 5). To assess the (http://www.xuru.org/st/ DS.asp). completeness of the assembled data, the assembled transcripts were To assess the de novo-assembled sequence of selected bphCad- analyzed using the arthropod gene sets in the BUSCO database as herins, bphAPNs, and bphALPs, RT-PCR was conducted using reference by the BUSCO program (Simão et al. 2015). The results Premix Taq (Ex Taq Version 2.0 plus dye) (TaKaRa, Japan) with the showed that out of 2,675 BUSCO searched genes, 84.11% (2,250 midgut cDNA as the template. Primer pairs used for PCR reactions BUSCOs) were complete, 5.08% (136 BUSCOs) were complete and were designed based on the assembled unigene sequences and are duplicated, 4.49% (120 BUSCOs) were fragmented, and 11.40% shown in Supp Table 2. The PCR fragments were isolated from the (305 BUSCOs) were not found, suggesting that the quality of the agarose gel and shotgun sequenced to verify the de novo-assem- assembly was completely acceptable. The ORF sequences were pre- bled sequence of selected bphAPNs, bphALPs, and bphCadherins. dicted from the resultant unigenes by using the OrfPredictor pro- Verified sequences were submitted to the GenBank. The accession gram (Min et al. 2005). The numbers and the average length of the number of each gene is shown in Supp Table 3. predicted ORF sequences have been summarized in Supp Table 5. The transcriptome sequencing data set has been deposited in NCBI as BioProject ID PRJNA383084. Identification of Homologs of Cry Receptors and Annotation of the assembled transcripts was conducted by using Phylogenetic Analysis the BLAST program against the NCBI nr database with a cutoff To obtain the homologs of Cry receptor proteins expressed in the −5 E-value of 10 . Only 18,810 of 53,502 (35%) unigenes hit the ref- midgut of BPH, unigenes annotated with the keyword ‘cadherin’, erence genes from the NCBI nr database (Supp Table 6). Statistics ‘alkaline phosphates’ or ‘aminopeptidase N’ were screened through from the BLAST results (E-value, sequence similarity and hit species custom Perl script from all NCBI nr-annotated unigenes. The full- distributions) are shown in Supp Fig. 1. length genes were determined if the predicted ORF was inside the unigene sequence, together with the start codon, stop codon, 5ʹ-UTR Identification of Cadherin, APN, and ALP Genes and 3ʹ-UTR. Cry-binding cadherins, APNs, and ALPs in Cry tox- To identify putative homologous genes of Cry-binding cadherin, in-susceptible insects and aphids used for sequence identity, sequence APN, and ALP, we searched for the transcripts that hit cadherins, alignment, and phylogenetic analysis were randomly selected from APNs, and ALPs from the BLAST annotation and identified eight the GenBank. Protein alignment was performed with the MEGA 6.06 putative cadherins, five putative APNs, and six putative ALPs, software (Tamura et al. 2013) using the built-in MUSCLE program. respectively. Among those identified genes, one APN and two ALPs The poorly aligned positions and divergent regions of the alignment had been previously reported but with slight sequence variations were eliminated by the Gblocks program (Castresana 2000, Talavera (Supp Table 1). We then conducted RT-PCR to verify sequences of and Castresana 2007) to obtain conserved blocks. A sequence iden- the assembled genes that hit CBPs. The cDNA fragments amplified tity matrix was calculated by BioEdit 7.0.9. The multiple sequence by RT-PCR matched the expected lengths of the assembled sequences alignments and phylogenetic trees (maximum-likelihood trees) were that were tested (Supp Figs. 2–4). The assembled sequences were then generated using the MEGA 6.06 with a bootstrap value of 1000. confirmed by Sanger sequencing of the PCR fragments with only The amino acid sequence of a selected BPH protein was input a few nucleotide variations compared to the assembled sequences to SMART (http://smart.embl-heidelberg.de) for motif scanning. (results not shown). Prediction of a signal peptide at the N-terminus of each protein was conducted with SignalP 4.1 (Petersen et al. 2011). GPI-anchor signal at the C-terminus was predicted using PredGPI, FragAnchor, and Comparison of the CBPs With Their Putative big-PI Predictor. N-glycosylation and O-glycosylation sites were pre- Homologs in BPH dicted by the NetNglyc 1.0 Server (Blom et al. 2004) and NetOglyc To identify Cry receptor homologs in the BPH midgut, we first 4.0 Server (Julenius et al. 2005), respectively. Transmembrane helices analyzed the sequence similarity between the known Cry receptors of each protein were predicted by the TMHMM 2.0 Server (http:// derived from Bt-susceptible insects and their potential counterparts www.cbs.dtu.dk/services/TMHMM/). in BPH. We then analyzed putative motif regions and compared sequence variations of the motifs between Cry receptors and their Three-Dimensional Modeling of Selected APN potential homologs of BPH. and ALP Three-dimensional structure modeling of APN and ALP was based Sequence Analysis of the Putative BPH Cadherin, APN, and ALP on templates 4wz9.1.A and 1zef.1.A, respectively. Models were built Proteins by online software SWISS-MODEL (Arnold et al. 2006, Guex et al. Comparisons of protein sequence identities between Cry recep- 2009, Kiefer et al. 2009, Biasini et al. 2014) and displayed by the tor-related cadherins, APNs, and ALPs derived from lepidopteran, PyMOL Molecular Graphics System, Version 1.8, Schrödinger, LLC. coleopteran, and dipteran insects and those of BPH are summarized in Supp Tables 7–9, respectively. Overall, sequence identities of the previously reported Cry-binding cadherins were around 50% and Results even over 70% among insects of the same order. The only exception Sequencing, de novo Assembly, and Functional was bmCadherin2, which had a 36.5 and 31.1% identity with the Annotation known Cry-binding cadherins in B. mori (bmCadherin1) and T. ni Illumina sequencing of the midgut and whole-body transcriptome (tnCadherin), respectively (Supp Table 7). However, the sequence generated over 39 million and 33 million raw reads, respectively identities between the putative BPH cadherins and most of the (Supp Table 4). The raw reads of both midgut and whole body were selected Cry-binding cadherins were less than 30% (bphCadherin Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/10/4839024 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. XX, No. X 5–8) to ~40% (bphCadherin 1–4). Only bphCadherin1 showed over aphids. Interestingly, bphAPN1 showed distance from other APNs of a 50% sequence identity (53.8%) with a cadherin originating from hemipteran insects but was closer to the root of APNs from the vast B. mori (bmCadherin2) (Supp Table 7). The amino acid identities of majority of the lepidopteran and coleopteran insects (Fig. 1B). Similar APN proteins showed similar diversities (~35 to 40%) among the to APNs, the BPH ALPs were clustered with either ALPs of aphids or Cry-sensitive insects and between Cry target insects or nontarget ALPs of coleopterans (Fig. 1C). insects (BPH or aphids) (Supp Table 8). Compared to cadherin and APN, sequence identity of ALP isolated from BPH and Bt-susceptible Domain Characterization of the Selected Cadherin, APN, and insects was relatively higher (>50%) (Supp Table 9). ALP in the BPH Midgut To conduct phylogenetic analysis, the BPH proteins (bphCadherins, Signal peptide and transmembrane helices are essential sequence bphAPNs, and bphALPs) which showed higher sequence identity with components of the transmembrane proteins. In the four selected those of the Bt-susceptible insects were selected for construction of phy- bphCadherins, signal peptides were predicted from bphCadherin1, logenetic trees. It is not surprising that the protein sequences derived 2, and 3 by an online program SignalP (http://www.cbs.dtu.dk/). The from the same species or the same insect orders were mainly clustered Cry-binding cadherins contain various cadherin repeats (CAs), a into the same lineages with some exceptions (Fig. 1). For example, most membrane-proximal region (MPR), a transmembrane domain (TM), of the cadherins from moths, mosquitoes, and beetles were grouped and a cytoplasmic domain (CD) (Zhang et al. 2012). Prediction of based on orders of the insects, while bphCadherins and apCadherins of transmembrane helices through TMHMM suggested that all the hemipteran insects showed phylogenetic distance from the Cry-binding selected bphCadherins contain at least one transmembrane helix. In cadherins. However, a cadherin derived from Tribolium castaneum addition, more than 10 potential N-glycosylation sites and various (Coleoptera: Tenebrionidae) (tcCadherin2) and bmCadherin2 was numbers of O-glycosylation sites in each bphCadherin were pre- grouped together with bphCadherins and apCadherins. Sequence iden- dicted (Table 1). We also conducted motif scanning of the bphCad- tities of bmCadherin2 to apCadherins were up to 80.3% (Fig. 1A). But herins. The results suggested that only bphCadherin1 contained 14 bmCadherin2 and tcCadherin2 were not found to be associated with CAs, an MPR, a TM, and a CD, which is similar to the motifs found the insecticidal activity of Cry toxins. Phylogenetic analysis showed in Cry-binding cadherins (Fig. 2). Illustrations of the signal peptides, that the five deduced bphAPNs were either clustered with two coleop- 14 CAs, and TM of bpCadherin1 as examples of the analysis are teran APNs or grouped into the same lineage of the APNs derived from shown in Supp Fig. 5. Fig. 1 Molecular phylogenetic analysis of selected cadherins (A), APNs (B), and ALPs (C) from BPH transcriptome by maximum-likelihood method. Amino acid sequence alignment for each analysis was conducted by the MEGA 6.06 built-in MUSCLE program followed by the screening of conserved blocks by the Gblocks program. The evolutionary history was inferred by using the maximum-likelihood method based on the Le_Gascuel_2008 model (Le and Gascuel 2008). The bootstrap consensus tree inferred from 1,000 replicates (Felsenstein 1985) is taken to represent the evolutionary history of the taxa analyzed (Tamura et al. 2013). Initial trees for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using a JTT model. A discrete gamma distribution was used to model evolutionary rate differences among sites [four categories (+G, parameter = 3.2778)]. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA 6.06. Information about amino acid sequences included in each tree is shown in Additional Table 9. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/10/4839024 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. XX, No. X 5 Similar to bphCadherins, a signal peptide in the N-terminus of Tenebrio molitor (Coleoptera: Tenebrionidae) to identify putative bphAPN1, 2, and 4 was predicted (Supp Fig. 6). However, a puta- homologous domains. The results of sequence alignment suggested tive transmembrane helix was only found in bphAPN3 (Table 1) but that the CA12-14 and MPR of bphCadherin1 were homologous to not from bphAPN1, 2, and 4. The GPI-anchored sites are common some CBR domains of the Cry-binding cadherins (Fig. 2). Majority motifs found in Cry-associated APNs. Analysis of GPI-anchored of the highly conserved (≧80% conserved) amino acids among Cry- signals of bphAPNs by using three online prediction programs binding cadherins could be identified from CA12-14 in bphCad- (PredGPI, FragAnchor, and big-PI Predictor) suggested that only near herin1, while no obviously conserved site was found by comparing the C-terminus of bphAPN4 is there a high probability of finding a MPR of bphCadherin1 to its counterparts of Cry-binding cadherins GPI-anchored site (Supp Fig. 6; Table 1). In addition, both N- and (Fig. 2). There are varied numbers of CBRs in the Cry-binding cad- 865 875 O-glycosylation sites were predicted in five deduced bphAPNs with herins of different insects. Two small CBRs ( NITIHITDTNN 1331 1342 the bphAPN4 containing the most abundant N- and O-glycosylation and IPLPASILTVTV ) were found in the Cry-binding cadherin sites (18 N-linked glycosylation sites and 26 O-linked glycosylation of M. sexta (Zhang et al. 2013). Homologous sequences of these sites) (Table 1). All predicted N- or O-glycosylation sites are illus- two CBRs were mapped to the CA8 and CA13 regions of bphCad- trated in Supp Fig. 6. herin1, respectively. However, low identity between both CBRs of Analysis of the bphALPs detected a signal peptide at the Cry-binding cadherins and the corresponding regions in bphCad- 906 908 910 N-terminus from all bphALPs except for bphALP2 and 5 (Table 1). herin1, although three conserved amino acids ( I, D, and N) The transmembrane helices were only predicted from bphALP2 were identified (Fig. 2). There are three amino acid residues, L, 1429 1430 (two sites) and bphALP5 (one site) by TMHMM (Table 1). In add- F, and Q, in Cry-binding cadherin of H. virescens (Xie et al. 1362 1582 ition, bphALP2 and bphALP4 were predicted as GPI-anchored 2005) and a R to L region in the cadherin of T. ni (Zhang proteins, while bphALP6 was a putative GPI-anchored protein et al. 2013, Badran et al. 2016), which showed particular importance (Table 1, Supp Fig. 7). Prediction of N- or O- glycosylation sites for toxin binding to Cry1A toxins. These regions were aligned to showed that all bphALPs contain at least two N-glycosylation sites CA14 and MPR in bphCadherin1. But very low identity was found except for bphALP5, while all six bphALPs contain 8–12 putative in these sites of bphCadherin1 compared to that of the Cry-binding O-glycosylation sites (Table 1). The predicted N- or O-glycosylation cadherins (Fig. 2). sites are highlighted in Supp Fig. 7. Sequence alignment of bphAPNs and bphALPs with their homol- ogous CBPs was also conducted to identify homologous regions of Comparison of the Protein Functional Domains previously reported CBRs. One Cry1A toxin-binding region was Cry-binding regions (CBRs) of Cry-binding cadherins, APNs, and found from bphAPNs. Compared to the N-terminus of CBR in Cry- ALPs have previously been identified (Nakanishi et al. 2002, Budatha binding APNs, a specific motif like TQFxxTxARxAFPCxDEP at the et al. 2007, Fernandez et al. 2009, Gómez et al. 2012, Kaur et al. C-terminus in bphAPN exhibited a greater similarity to Cry-binding 2014). The above sequence analysis suggests that bphCadherin1 APNs of susceptible insects (Fig. 3). Homologous modeling of the may contain homologous domains of the Cry-binding cadherins protein 3D structure showed that the structure of bphAPN1 is sim- derived from susceptible insects. The sequence of bphCadherin1 was ilar to that of the hvAPN1, which is a known receptor of Cry1Ac then aligned to previously reported Cry-binding cadherins derived toxin in the midgut of H. virescens (Fig. 4). In addition, the CBR from Manduca sexta (Lepidoptera: Sphingidae), Spodoptera exi- homologous region in bphAPN1 is also predicted to be exposed on gua (Lepidoptera: Noctuidae), Heliothis virescens (Lepidoptera: the surface of the protein (Fig. 4). ALPs are important Bt receptors Noctuidae), T. ni, Anopheles gambiae, Aedes aegypt (Diptera: in the midgut of Cry-susceptible insects (Perera et al. 2009, Wang 95 102 257 296 Culicidae), Alphitobius diaperinus (Coleoptera: Tenebrionidae), and 2015). Two CBRs ( R- G and N- I) were reported to play Table 1. Characters of potential Cry receptor in BPH Predicted proteins Signal P GPI-anchored sites Transmembrane helix† Number of Number of N-glycosylation site O-glycosylation site PredGPI fragAnchor big-PI Predictor bphCadherin1 ○ N/A N/A N/A o1687-1709i 15 2 bphCadherin2 ○ N/A N/A N/A i7-26o1369-1391i 14 2 bphCadherin3 ○ N/A N/A N/A o1376-1398i 10 9 bphCadherin4 × N/A N/A N/A o1579-1601i 17 3 bphAPN1 ○ × × × o 10 3 bphAPN2 ○ × × × o 6 2 bphAPN3 × × × × i33-55o 7 5 bphAPN4 ○ ○ ○ × o 18 26 bphAPN5 × × × × o 4 1 bphALP1 ○ × × × o 3 11 bphALP2 × ○ ○ ○ i12-34o508-530i 2 8 bphALP3 ○ × × × o 3 12 bphALP4 ○ ○ ○ ○ o 3 9 bphALP5 × × × × i7-29o 0 8 bphALP6 ○ ○ × × o 2 11 N/A indicates character prediction was not conducted. † i and o in the row of transmembrane helix indicated the domain inside and outside the cell membrane, respectively. Numbers indicate the starting amino acids of domains. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/10/4839024 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. XX, No. X Fig. 2 Overview of sequence components in bphCadherin1 and multiple alignments of specific motifs in bphCadherin1 against Cry receptor cadherins. The signal peptide (Signal P), transmembrane domain (TM), membrane proximal region (MPR), cytoplasmic domain, and numbered cadherin repeats (CAs) are illustrated. The sequence of each CA containing previously reported Cry-binding regions is shown as insets. Amino acids equal to or greater than 50% conserved are shaded in blue while those 80% conserved are shaded in red. Fig. 3 Schematic presentation of sequence components in bphAPNs and multiple alignments of specific motifs in bphAPNs against Cry receptor APNs. Predicted positions of signal peptide and GPI-anchored sites are boxed in red at N- and C-terminuses, respectively. Cry-binding region (CBR) and the conserved GAMEN 2+ and gluzincin sequences involved in Zn binding are boxed in blue, yellow, and green, respectively. Insets show the detailed alignment of each motif between amino acid sequences of bphAPNs and Cry receptor APNs. Amino acids equal to or greater than 50% conserved are shaded in blue while those 80% conserved are shaded in red. essential roles in mediating interaction of Cry-binding ALPs with Relative Transcript Abundance of Putative Cry Cry toxins (Fernandez et al. 2009). Homologous sequences of the Receptor Homologous Genes in the BPH Midgut two CBRs were identified in bphALPs (Fig. 5), which showed a sim- Relative expression of bphCadherins, bphAPNs, and bphALPs in the ilar surface-exposed 3D structure to that of the Cry-binding ALP midgut of BPH were estimated by per kilobase of exon model per in H. virescens (Fig. 6). In addition, a highly conserved region was million mapped reads (RPKM), which showed that transcript abun- found in the C-terminus of CBR1 and its homologous regions of dance of bphAPN1 and bphAPN4 was significantly greater than that bphALPs, while the sequences of CBR2 homologs of bphALPs were of other APNs in the midgut of BPH. The highest RPKM of ALP in closer to that of Cry-binding ALPs at the N-terminus (Fig. 5). the BPH midgut was bphALP6 and bphALP4. To verify the rela- In addition to the CBRs, specific motifs were also predicted from tive transcript abundance determined by transcriptome sequencing, BPH APNs or ALPs. A gluzincin aminopeptidase motif (GAMEN) RT-qPCR was conducted to examine selected BPH APN and ALP and a zinc-binding/gluzincin motif (HEXXHX E), which are expressions, which confirmed the results calculated from the tran- highly conserved in APNs, were detected from the sequence of five script reads (Table 2). Compared to APNs and ALPs, bphCadherins deduced bphAPNs (Supp Fig. 6). Both motifs show high sequence were expressed at a low to moderate level in the midgut (Table 2). conservation compared to the reported APNs of lepidopteran, coleopteran, dipteran insects, and aphids (hemipterans) (Fig. 3). Discussion Similarly, as typical characteristics of ALPs, phosphatase-active sites and a phosphatase-active domain (Perera et al. 2009), which Cry-receptor interaction is necessary for Cry toxin insecticidal showed high sequence similarity to the ALPs of Bt-susceptible activities to occur. It was observed that the deletion of putative insects and the soybean aphid, were predicted in all six bphALPs Cry-receptor binding sites or low expression of specific receptor (Fig. 5 and Supp Fig. 7). protein could significantly reduce the susceptibility of susceptible Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/10/4839024 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. XX, No. X 7 Fig. 4 Three-dimensional structures of hvAPN1 and bphAPN4 were respectively constructed based on template 4wz9.1.A in the SWISS-MODEL template library. The surface structure of APN proteins was predicted by built-in Python script of the PyMOL Molecular Graphics System. CBR residues in either hvAPN1 or bphAPN4 were displayed as spheres, while the rest of the amino acids were displayed as surface structures. Surface residues in CBR were stained in green. Surface residues not included in CBR were stained in red. The top panel indicates the sequence alignment between CBRs. Fig. 5 Schematic presentation of sequence components in bphALPs and multiple alignments of specific motifs in bphALPs against Cry-receptor ALPs. Predicted positions of signal peptide and GPI-anchored sites are boxed in red at N- and C-terminuses, respectively. Cry-binding regions (CBRs) and the conserved alkaline phosphatase active domain (ALPAD) are boxed in blue and green, respectively. Insets show the detailed alignment of each motif between amino acid sequences of bphALPs and Cry-receptor ALPs. Amino acids equal to or greater than 50% conserved are shaded in blue while those 80% conserved are shaded in red. insects to the related Cry toxins (Wang 2015). In addition, rein- between Bt toxins and hemipteran insect guts could possibly retar- stallation of the dysfunctional receptor-binding domains of the get the toxins against hemipteran insects. Hence, identification of resistant insect strains could restore the toxin insecticidal activ- Bt-toxin receptor homologous proteins in BPH midguts and com- ity against the previously resistant insects (Badran et al. 2016). parison of the differences with previously reported Bt-toxin recep- Studies on replacements of Bt toxin (Cry or Cyt) functional tors may provide valuable information in understanding the mode domains with gut-binding peptide (GBP) of aphids and BPH of action of Bt toxins in BPH. revealed that GBP improved the toxin-midgut binding affinity and In this report, we have investigated the homologous genes of resulted in a significant increase in toxin activity against the aphids Cry receptors (cadherin, apn, and alp) from the transcriptome of and BPH (Chougule et al. 2013, Shao et al. 2016). These results N. lugens nymphs and then analyzed the protein sequence, func- demonstrated that the insecticidal activity of Cry toxins was firmly tional domains, protein structures, and transcript abundance of the related to toxin-receptor binding. Improvement of binding affinity related genes. We have identified at least one cadherin, one APN, and Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/10/4839024 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8 Journal of Insect Science, 2018, Vol. XX, No. X Fig. 6 Three-dimensional structures of hvALP1, bphALP2, and bphALP4 were respectively constructed based on template 1zef.1.A in the SWISS-MODEL template library. The surface structure of ALP proteins was predicted by built-in Python script of the PyMOL Molecular Graphics System. CBR residues in bphALP2, bphALP4, and hvALP1 were displayed as spheres, respectively, while the rest of the amino acids were displayed as surface structures. Surface residues in CBR1 and CBR2 were stained in green and cyan, respectively. Surface residues not included in CBR were stained in red. The panels on the top and bottom indicate the sequence alignment of CBR1 and CBR2, respectively. BPH, while a low to moderate expression level of all cadherins was Table 2. Relative transcript abundance of selected homologs of detected in the transcription level (Table 2). The transcription abun- Cry receptor proteins in the midgut of BPH dance of bphCadherin, bphAPN, and bphALP was similar to that of Deduced gene RPKM Relative abundance (△CT±SD) their homologs in the midgut of Bt-susceptible insects (Tiewsiri and Wang 2011, Zhang et al. 2012, Chen et al. 2015). Cry-binding cad- bphcadherin1 5.59 6.68 ± 0.86 herins, APNs, and ALPs were known as membrane-bound proteins bphcadherin2 20.39 7.41 ± 0.94 (Wang 2015). Prediction of transmembrane helix and GPI-anchored bphcadherin3 3.7 5.45 ± 0.75 signal showed that bphAPN4, bphALP2, bphALP4, bphALP6, and bphcadherin4 0.69 2.08 ± 0.89 bphAPN1 1182.63 10.88 ± 0.23 all bphCadherins are potentially membrane-bound proteins with bphAPN2 5.05 3.19 ± 0.37 either GPI-anchor signal or transmembrane helix (Table 1). Hence, bphAPN3 2.00 3.18 ± 0.45 it is possible that the bphCadherins, bphAPNs, and bphALPs have bphAPN4 1263.03 10.25 ± 0.24 features similar to their homologs in Bt-susceptible insects. bphAPN5 10.39 5.39 ± 0.50 The results of sequence alignment indicated that bphAPN4, bphALP1 0.29 1.08 ± 0.34 bphALP2, and bphALP4 shared moderate to high protein identity bphALP2 9.59 1.45 ± 0.41 with their CBP homologs (Supp Tables 7–9), although the pro- bphALP3 0.59 0.76 ± 0.52 tein sequences of bphCadherins, bphAPNs, and bphALPs were bphALP4 302.37 8.67 ± 0.29 highly divergent compared to those of the CBPs in Cry-susceptible bphALP5 2.06 1.70 ± 0.41 insects (Fig. 1). In addition, bphCadherin1, bphAPN4, bphALP2, bphALP6 2020.66 11.67 ± 0.22 and bphALP4 contained domains similar to the Cry-binding domains of the CBPs. However, it is not surprising to observe that Reads Per Kilo base per million mapped reads (RPKM) of was calculated to predict transcript abundance of putative Bt receptor genes. The relative the majorities of the amino acids in these domains differed from abundance (△Ct) of selected APNs, ALPs, and cadherins in the midgut was CBPs (Figs. 2, 3, 5). Studies on the Cry-cadherin interaction indi- 865 875 1331 1342 normalized using the BPH 18s rRNA threshold cycle (Ct) values that were cated that CBRs NITIHITDTNN and IPLPASILTVTV obtained from reactions run on the same plate. in the Cry-binding cadherin of M. sexta and repeat 12 in the Cry- binding cadherin of H. virescens could interact with the domain II two ALP proteins that showed similar characteristics to the previ- loop 2, loop α8, and loop 3 of Cry toxins, respectively (Bravo et al. ously reported CBPs in lepidopterans, coleopterans, and dipterans. 2007). Sequence alignment showed that nearly no sequence simi- Cry-binding APNs and ALPs were reported to have a high expres- larity between the corresponding regions in bphCadherins and the sion level in the midgut of susceptible insects, while cadherin showed three CBRs of the lepidopteran Cry-binding cadherins. Cry recep- a relatively low abundance level (Chen et al. 2005, 2009). Results tor-related APN and ALP usually contain a GPI site and numbers of of RPKM calculation and RT-qPCR assay showed that bphAPN1, N- or O-glycosylation sites. Glycosyl-mediated interaction between bphAPN4, and bphALP6 were highly abundant in the midgut of Cry toxins and GPI-anchored APN or ALP was reported, suggesting Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/10/4839024 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. XX, No. X 9 Bravo, A., I. Gómez, H. Porta, B. I. García-Gómez, C. Rodriguez-Almazan, that these glycosyl sites could be important in the mechanism of Cry L. Pardo, and M. Soberón. 2013. Evolution of Bacillus thuringiensis Cry insecticidal activity (Luo et al. 1999, Shinkawa et al. 1999, Fernandez toxins insecticidal activity. Microb. Biotechnol. 6: 17–26. et al. 2009, Lin et al. 2014). The bphAPN4, bphALP2, and bphALP4 Budatha, M., G. Meur, and A. Dutta-Gupta. 2007. A novel aminopeptidase were predicted to contain numbers of N- and O-glycosylation sites in the fat body of the moth Achaea janata as a receptor for Bacillus thur- and a GPI-anchored site, suggesting that bphAPN4, bphALP2, and ingiensis Cry toxins and its comparison with midgut aminopeptidase. bphALP4 are potential to interact with Cry toxins. However, CBRs Biochem. j. 405: 287–297. of bphAPN and bphALP are divergent to the Cry-binding APNs Castresana, J. 2000. Selection of conserved blocks from multiple alignments and ALPs. Consequently, high divergence of Cry-binding cadherins, for their use in phylogenetic analysis. Mol. Biol. Evol. 17: 540–552. APNs, and ALPs with their homologs in BPH may be part of the Chen, J., M. R. Brown, G. Hua, and M. J. Adang. 2005. Comparison of the reason why BPH is not susceptible to Cry toxins. localization of Bacillus thuringiensis Cry1A delta-endotoxins and their binding proteins in larval midgut of tobacco hornworm, Manduca sexta. Improper binding of Cry toxins in the midgut of BPH and pea Cell Tissue Res. 321: 123–129. aphid has been known to be a possible reason for the low toxicity of Chen, J., K. G. Aimanova, L. E. Fernandez, A. Bravo, M. Soberon, and S. these toxins (Li et al. 2011, Shao et al. 2013, Niu et al. 2017). Our S. Gill. 2009. Aedes aegypti cadherin serves as a putative receptor of the results provide not only sequence characteristics but also structural Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis. Biochem. j. characteristics of the CBP-like proteins of BPH. Information from 424: 191–200. this study may also enrich our knowledge of the mode of action of Chen, W., C. Liu, Y. Xiao, D. Zhang, Y. Zhang, X. Li, B. E. Tabashnik, and Bt toxins in the midgut of hemipteran insects. K. Wu. 2015. A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera. PLoS One. 10: e0126288. Supplementary Data Chougule, N. P., and B. C. Bonning. 2012. Toxins for transgenic resistance to hemipteran pests. Toxins (Basel). 4: 405–429. Supplementary data are available at Journal of Insect Science online. Chougule, N. P., H. Li, S. Liu, L. B. Linz, K. E. Narva, T. Meade, and B. C. Bonning. 2013. Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests. Proc. Natl. Acad. Sci. USA. 110: Acknowledgments 8465–8470. We thank Connie Allison for critical reading and editing of the manuscript. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the We also thank Institute of Plant Virology, FAFU, for providing us the wild-type bootstrap. Evolution. 39: 783–791. N. lugens strain. This study was supported by Project of Fujian-Taiwan Joint Fernandez, L. E., C. Martinez-Anaya, E. Lira, J. Chen, A. Evans, S. Hernández- Center for Ecological Control of Crop Pests (grant number Minjiaoke 2013- Martínez, H. Lanz-Mendoza, A. Bravo, S. S. Gill, and M. Soberón. 2009. 51); National Natural Science Foundation of China (grant number 31401802 Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa- and 31772539); Science and Technology Project in Fujian Province Department receptor alkaline phosphatase from Aedes aegypti. Biochemistry. 48: of Education (grant number JA15161); Research development fund of Fujian 8899–8907. Agricultural and Forestry University (grant number KF2015041) and Science and Gómez, I., L. Pardo-López, C. Muñoz-Garay, L. E. Fernandez, C. Pérez, J. 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