Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella
© Tang et al.; licensee BioMed Central Ltd. 2012
Received: 13 June 2012
Accepted: 11 October 2012
Published: 19 October 2012
The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors.
In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro- translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM). In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous.
Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists) to their targets (ecdysone receptors) leads to an adaptive response (resistance), is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.
KeywordsPlutella xylostella, Ecdysone receptor (EcR) Binding affinity, Expression profiling, Ecdysone agonist
Ecdysteroids, a family of lipophilic poly-hydroxylated steroids, are hormonal factors modulating a diverse array of physiological processes such as development, reproduction, homeostasis and metabolism in arthropoda, especially in insects[1, 2]. The proper developmental transitions in insects, including molting and metamorphosis, demand accurate cellular synthesis and reorganization and precise timing/programming of gene expression associated with various stages of development. A classic example of such programming is reflected in the study of Manduca sexta (Lepidoptera: Sphigidae) by Lynn Riddiford and her colleagues[5, 6]. In Lepidoptera, juvenile hormone (JH) is present rhythmically prior to the induction of ecdysteroids to orchestrate the precise developmental plan for insects. 20-hydroxyecdysone (20E), is one of the most common and important ecdysteroids. The actions of 20E are evidently mediated by the ecdysone receptor complex, a heterodimer of two members of the nuclear receptors superfamily (NRs), the ecdysteroid receptor (EcR), and the ultraspiracle (USP).
Structural features in these heterodimeric complexes shed lights on how ecdysteroids initiate molting and metamorphosis in insects. As reviewed by Nakagawa and Henrich, both EcR and USP exhibit a basic structure of typical NRs, commonly including 4-5 domains, namely the A/B, C, D, E, and in some receptors, F. The A/B domain at amino terminal is extremely variable, which contains a ligand-independent transcriptional activation function 1(AF-1), and interacts with other transcriptional factors. The C domain, the central DNA-binding domain (DBD), contains two highly conserved zinc finger motifs that are characteristic of the nuclear receptor superfamily (NRs). The D domain, a more variable region, is referred to as a hinge region between the C and E regions and harbors nuclear localization signals. It was reported by Graham et al. that the D domain could influence the binding of ligand to the receptors in Helicoverpa armigera. The E domain, referred to as the ligand binding domain (LBD), functions uniquely to NRs and is well-conserved. For EcR, the LBD plays roles in receptor dimerization, ligand recognition and cofactor interactions. Recently, the flexible ligand-dependent binding pocket where steroidal and non-steroidal binds, has been characterized by X-ray crystallography[10–13]. The F domain is divergent and its functions are still unknown.
The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a highly destructive pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including the molt-accelerating compounds/ ecdysone agonists, such as diacylhydrazine (DAH) insecticides[15, 16]. DAH-based biopesticides have been used to control various agriculture, forestry, and stored product pests for the past decade[17–19], and been considered an environmentally friendly insecticide because of their remarkable selectivity across taxonomic orders, especially their compatibility with predatory biological control agents. DAHs function by binding to the ecdysone receptor complex to compete with ecdysteroids, and to interfere with genes involved in the cuticle secretion to induce a lethal precocious incomplete molt, especially in Lepidoptera[21, 22]. Previously we reported that the catabolism of ecdysteroid agonists (e.g., Fufenozide, a non-steroidal ecdysone agonist) may play a major role in the acquisition of fufenozide resistance in P. xylostella. Other potential resistance mechanisms, such as the signaling of ecdysteroid receptor complex, involving both EcR and its heterodimer partner receptor USP, have yet been investigated.
As the eventual target of ecdysone agonists/molt-accelerating biopesticides, insect ecdysone receptors have been extensively studied in Diptera and Lepidoptera. In P. xylostella, an agriculturally important insect pest, ecdysone receptors have not been documented except EcRB (EF417582). In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including both EcR and USP. The binding affinity of ecdysteroids to in vitro - translated receptor proteins was investigated. Moreover, the mRNA expression profiles of EcRs and USP, respectively, across different developmental stages were also documented. These combined results shed light on the molecular understanding of mechanisms underlying the ecdysone agonist resistance in P. xylostella.
Insects and RNA isolation
Larvae and adults of P. xylostella were maintained at 27 ± 1°C, 70 ± 10% RH, and a 16:8 L: D photoperiod, as described previously. Total RNA was isolated from the whole body homogenates of the last-instar larvae (4th), pupae and adult females using TRIzol reagent (Invitrogen, Carlsbad CA, USA) following the manufacturer’s instructions. The concentration and purity of the total RNA were determined using a Thermo scientific NanoDrop 2000.
Reverse transcription polymerase chain reaction (RT-PCR)
Reverse-transcription was conducted using PrimeScript 1st strand cDNA synthesize kit (Takara Biotechnology Co., Ltd, Dalian, China). For the cloning of PxEcRB (GenBank accession number: EF417582), specific primers (Additional file1: Table S1) were designed and the PCR was performed with GC buffer Ι and LA Taq (Takara) as follows: 94°C/ 4 min; 30 cycles of 94°C /45 s, 57.8°C/40 s, 72°C/ 2 min; and 72°C/10 min. For PxUSP, degenerate primers (Additional file1: Table S1) were designed based on amino acid sequences conserved in the C and E regions of other lepidopterean USPs, and the PCR was conducted as follows: 94°C/4 min; 30 cycles of 94°C/ 40s, 57.8°C/ 40s, 72°C/1 min20s; and 72°C/10min.
Rapid amplification of cDNA ends (RACE)
Given that the isoform-specific A/B domains, the 5’-RACE primers for both USP and EcR were designed in the C region, and one reverse primer for 5’-RACE and one forward primer for 3’-RACE were designed, respectively (Additional file1: Table S1). Total RNAs from adult females, pupae and the 4th instar larvae, respectively, were subjected to 5’-RACE with Smart™ Race cDNA Amplification Kit (Clontech, Palo Alto CA, USA) according to manufacturer’s instructions. The cycles at annealing temperature of 68°C was 30 instead of 25. For 3’-RACE of USP, total RNA from the last-instar larvae was subjected to 3’-Full RACE Core Set Ver.2.0 (Takara) according to manufacturer’s instructions.
PCR products were purified by agarose gel electrophoresis and cloned into the pGEM-T Easy vector (Promega, Madison WI, USA) before submission to Invitrogen (Shanghai, China) for sequencing. cDNA sequence, deduced amino acid sequences, and multiple sequence alignments were analyzed using DNAMAN 5.2 program. Sequence similarity of each domain imbedded in EcR and USP, respectively, was calculated by BLAST. Phylogenetic relationships of ecdysone receptors from P. xylostella with other insects were analyzed using CLUSTAL X 2.0 and MEGA 5.0 based on their amino acid sequences. Both NJ (neighbor-joining, model: poisson-correction, bootstrap values: 1000 replicates) and ML (maximum likelihood, model: Jones Taylor Thornton (JTT), bootstrap values: 500 replicates) trees were constructed and compared. All protein sequences were acquired from the GenBank.
In vitro transcription-translation
Complete opening reading frames (ORFs) of PxEcRA, PxEcRB and PxUSP were amplified using primers listed in Additional file1: Table S1 with LA Taq and cloned, respectively, into pF25 T7 Flexi Vector (Promega), which can act as an acceptor of a protein-coding region flanked by Sgf I and Pme I sites. In vitro transcription-translation of these constructs was carried out using TNT T7 Insect Cell Extract Protein Expression system (Promega) according to manufacturer’s protocol. To validate the expression, proteins were synthesized in the presence of Transcend™ tRNA (a precharged, ε-labeled biotinylated lysine-tRNA complex, Promega) and separated on a 10% SDS–PAGE gel. After electro-blotting, the biotinylated proteins were visualized by binding Streptavidin-Alkaline Phosphatase, and followed by the colorimetric detection.
To compare the binding properties of the two EcR isoforms and to investigate their interactions with USP, the radioactive ligand-binding assay was performed according to Minakuchi et al. and Graham et al., respectively[9, 27]. In vitro translated proteins were diluted 1:2 with low-salt buffer (20 mM HEPES, 20 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.9, containing 1μg·ml-1 of aprotinin, pepstatin, leupeptin and 0.5 mg·ml-1 of bovine serum albumin). Diluted proteins (4 μl EcR, USP or a mixture of both) were incubated with 10 nM 3H-Labeled ponasterone A (tritiated PonA, 120 Ci·mmol-1, PerkinElmer Inc. Shelton CA, USA) in silicon tubes for 90 min at 25°C. No heterodimer protein controls (in vitro translation reaction prepared with an empty expression vector rather than a receptor) were regarded as the non-specific binding. The total volume of the reaction mixture was 16 μl, and the final concentration of solvent (ethanol) was less than 1%. At the end of the incubation, the samples were placed on ice and filtered immediately through nitrocellulose membrane (NC45, Merk Milipore) under a vacuum filtration apparatus. The membrane was then washed 3 times with 3ml of ice-cold washing buffer (low salt buffer with no protease inhibitors and BSA). Air-dried for ~10s, the membrane was transferred into a glass vial containing 2 ml of Aquasol-2 (PerkinElmer Inc.) in an oscillator for solvating. The radioactivity was then measured using a liquid scintillation counter Hidex-300SL (2 min/filter, Hidex instrument, Finland). In saturation binding experiments, proteins were incubated with 5 concentrations of tritiated PonA (0.625, 1.25, 2.5, 5.0 and 10 nM). Assays were performed in duplicate. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were caculated through non-linear regression using SigmaPlot 11.0 (Systat Software Inc., San Jose CA, USA).
Quantitative real-time PCR (qRT-PCR)
Gene expression profiles of PxEcRs and PxUSP, respectively, were examined throughout the fourth instar and in different developmental stages. For expressions within the fourth instar, the time of newly exuviated fourth instar was assigned as 0 h and RNA samples were collected at 6 h intervals untill pre-pupation. For expression profiles across different developmental stages, the total RNAs were isolated from a mixtures of individuals developed at different time intervals, i.e., the RNA samples for the 3rd instar larvae were collected at 0, 12, 24 and 36h. Besides, adult samples were kept in male to female ratio of 1:1. Total RNA was extracted as described above and treated with DNase I (Takara), and then 1μg of total RNA was subjected to the PrimeScript 1st strand cDNA synthesize kit (Takara). The qRT–PCR was conducted using Platinum SYBR Green qPCR SuperMix-UDG Kit (Invitrogen) with a 20 μl reaction volume containing 250 nM primer and 100ng of cDNA in an ABI 7300 System. Ribosomal protein L32 of P. xylostella (PxL32) was used as a reference gene, and specific primers (Additional file1: Table S1) for receptors were designed in the isoform-specific A/B domain using a web-based primer design platform, Primer 3 (http://frodo.wi.mit.edu/primer3/). Standard curves of each gene were prepared, respectively, via serial dilutions (10×) of cDNA samples. The optimized qPCR profile was as follows: 50°C/2min, 95°C/2min, 50 cycles of 95°C/ 15s, 60°C/ 30s, and with a dissociation step. All calculations were carried out by the accompanying softwares of ABI 7300 System, and statistical analysis was conducted by one-way ANOVA and Tukey’s test using GraphPad InStat (GraphPad Software Inc., San Diego CA, USA).
Molecular cloning of EcR and USP
In vitro translation of PxEcRs and PxUSP
Specific binding of tritiated PonA to in vitro-translated protein
Developmental expression profiles of PxEcRs and PxUSP
In this study, we cloned and expressed the full length cDNAs of three ecdysone receptors including two EcR isoforms and one USP from P. xylostella. PxEcRA was obtained from the adult female, whereas, PxEcRB was readily obtainable in each and every developmental stage. We also determined the expression profiles of their respective mRNAs in the 4th instar larvae as well as the entire developmental stages. Furthermore, when co-expressed with USP, the binding affinities of these EcR isoforms were examined.
Two EcR isoforms, PxEcRA and PxEcRB, differ in their A/B domain. These two isoforms belong to the Type 2 A and Type 6 B1 isoform, respectively, according to Watanabe et al.. The N-terminal A/B region of EcRA and EcRB is isoform-specific, and might be an essential structural basis for the transcriptional activation functions[2, 30, 31]. The (K/R)RRW motif of the EcR-B1 isoform provides additional interaction sites for co-regulatory proteins and mediates the regulation of the B1 isoform-specific AF1 transactivation function. In this study, for both PxEcRA and PxEcRB, two splicing variants with a 15 bp difference in the hinge domain were identified. A 15bp difference has also been reported in the splicing variants from M. sexta, C. suppressalis, and Leptinotarsa decemlineata. However, the functional significance of these structural differences between the two variants has yet been fully characterized. In C. suppressalis, however, the lack of five amino acids (encoded by 15 nt) in the middle of D region of EcR did not affect the ligand-receptor binding. Similar to EcRs, multiple USP isoforms have been found in lepidopteran M. sexta and H. armigera[33, 34], dipteran Aedes aegypti and Chironomus tentans[35, 36], and coleopteran Tribolium castaneum. In this study, however, only one USP isoform was obtained in P. xylostella. Other insect species containing only one USP isoform includes D. melanogaster, Chilo suppressalis and Choristoneura fumiferana.
Results from the ligand-binding assay demonstrated that 1) tritiated PonA specifically bound to PxEcRB but not PxUSP and PxEcRA, and the specific binding to PxEcRB was enhanced by the addition of PxUSP. This is consistent with previous observations in C. suppressalis, L. decemlineata, C. tentans[36, 41] and D. melanogaster; and 2) the presence of PxUSP could not enhance the binding of tritiated PonA to PxEcRA. Similar result was observed in the Dwarf Wood scorpion, Liocheles australasiae, where the retinoid X receptor did not enhance the binding of tritiated PonA to L. australasiae ecdysone receptor-A. Unlike C. fumiferana, where the two isoform complexes have similar binding affinities for ponasternone A, there is a distinct binding affinity between PxEcRA/USP and PxEcRB/USP.
The dissociation constant (Kd =3.0±1.7 nM) for the PxEcRB/USP heterodimer is comparable to EcR/USP complex from other insects, such as in lepidopteran 1.0-2.0 nM[27, 44, 45], coleopteran 2.8 nM for L. decemlineata, dipsteran 0.9-2.8nM[2, 46], pentatomomor-phan 6.8 nM for Nezara viridula, and orthopteran 1.2 nM for Locusta migratoria. Comparative analysis of receptor-binding affinity among insects sheds light on the divergence of the toxicity of ecdysone agonists.
Previous studies showed that the expression profiles of different isoforms vary among developmental stages and tissues. In D. melanogaster, EcRA isoform predominately locates at the imaginal discs, imaginal rings, and in two sets of specialized larval cells (the prothoracic gland cells). In addition, the EcRA isoform is associated with the onset of new cuticle synthesis in every molt so its presence is not metamorphic-specific. In contrast, the EcRB1 isoform is found primarily in larval tissues and in the imaginal histoblasts that form the abdominal epithelium and the midgut of adult[49, 50]. Phenotypic analysis of mutants suggests that EcRA is required for pupal development and EcRB1 is necessary for normal metamorphic development. In Bombyx mori, during the larval-pupal transition, EcRB1 was broadly distributed in most tissues examined including midgut, epidermis, fat body, and the wing imaginal disc, while EcRA was found only in the anterior silk gland. In this study, mRNA expression levels of PxEcRB were consistently 6-fold higher than that of PxEcRA in P. xylostella throughout the entire developmental stages. By contrast, the expression of EcRA in L. decemlineata was significantly higher than that of EcRB1, and EcRA was predominantly found in larval tissues. The apparent spatial and temporal distribution discrepancies of EcR isoforms are likely due to the different species.
This paper reports the molecular cloning, heterologous protein expression, and characterization of the three ecdysone receptors including two EcR isoforms and one USP from P. xylostella. As the target site of the DAH-based ecdysone agonist/molt-accelerating biopesticides, a comprehensive understanding of ecdysone receptors will shed light on the mechanistic study of DAH resistance in P. xylostella. The mode of action of this group of biopesticides is the induction of premature and lethal molting in insects. One of the resistance mechanisms involves altered binding of ecdysone agonists to the ecdysone receptor[53, 54], suggesting that target site insensitivity is the underlying resistance mechanism. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis, and more importantly, the potential role played by PxEcRB in diacylhydrazine resistance in P. xylostella warrants further investigation.
Nuclear receptor superfamily
Activation function 1
Nuclear localization signal.
This research is supported by grants from the National Natural Science Foundation of China (Nos. 30971941 and 31171873), the Special Fund for Agroscientific Research in the Public Interest (No. 201103021), the National 863 plans project (No. 2012AA101502). Special thanks go to John J. Obrycki (University of Kentucky) for his comments on an earlier draft.
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