Cry11Aa Interacts with the ATP-Binding Protein from Culex

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Cry11Aa Interacts with the ATP-Binding Protein from Culex quinquefasciatus To Improve the Toxicity Lingling Zhang,†,‡,§ Guohui Zhao,† Xiaohua Hu,† Jiannan Liu,† Mingwei Li,† Khadija Batool,† Mingfeng Chen,† Junxiang Wang,† Jin Xu,† Tianpei Huang,† Xiaohong Pan,† Lei Xu,† Xiao-Qiang Yu,*,†,§ and Xiong Guan*,†,‡ †

State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops and School of Life Science and ‡Fujian−Taiwan Joint Center for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, People’s Republic of China § Division of Cell Biology and Biophysics, University of MissouriKansas City, Kansas City, Missouri 64110, United States S Supporting Information *

ABSTRACT: Cry11Aa displays high toxicity to the larvae of several mosquito species, including Aedes, Culex, and Anopheles. To study its binding characterization against Culex quinquefasciatus, Cry11Aa was purified and western blot results showed that Cry11Aa could bind successfully to the brush border membrane vesicles. To identify Cry11Aa-binding proteins in C. quinquefasciatus, a biotin-based protein pull-down experiment was performed and seven Cry11Aa-binding proteins were isolated from the midgut of C. quinquefasciatus larvae. Analysis of liquid chromatography−tandem mass spectrometry showed that one of the Cry11Aa-binding proteins is the ATP-binding domain 1 family member B. To investigate its binding property and effect on the toxicity of Cry11Aa, western blot, far-western blot, enzyme-linked immunosorbent assay, and bioassays of Cry11Aa in the presence and absence of the recombinant ATP-binding protein were performed. Our results showed that the ATP-binding protein interacted with Cry11Aa and increased the toxicity of Cry11Aa against C. quinquefasciatus. Our study suggests that midgut proteins other than the toxin receptors may modulate the toxicity of Cry toxins against mosquitoes. KEYWORDS: ATP-binding protein, Bacillus thuringiensis, Cry11Aa, Culex quinquefasciatus, receptors

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INTRODUCTION Mosquitoes are an enormous public health menace in transmitting various tropical diseases, such as dengue fever, hemorrhagic fever, malaria, yellow fever, West Nile virus, chikungunya, Zika, etc.1,2 They are detrimental to the health and well-being of livestock and wildlife. Global health problems associated with mosquito-borne diseases put hundreds of millions of people at risk.3 For example, the recent outbreaks of Zika virus infections have captivated the global attention. Zika virus is transmitted primarily by Aedes mosquitoes, such as Aedes aegypti and Aedes albopictus.4,5 Culex mosquitoes mainly transmit filariasis and Japanese encephalitis. Filarial worms can parasitize in the human lymphatic system, causing lymphadenitis and lymphangitis and even lymphatic obstruction. Japanese encephalitis is a central nervous system disease caused by encephalitis virus.6,7 Without effective remedy and vaccine, mosquito management is an essential component for the control of mosquito-borne diseases by reducing its population density and cutting off the transmission of vector-borne diseases.3,5 Presently, destroying the breeding ground of mosquitoes, spraying chemical pesticides, and biological control are the main approaches for mosquito control. Protection of environmental sanitation and then destruction of the mosquito breeding ground are effective to reduce the mosquito population, but mosquitoes can reproduce and grow in other naturally wet and warm conditions quickly; thus, they are difficult to be eliminated completely. Although effective in © XXXX American Chemical Society

mosquito control, chemical pesticides have many defects, such as environmental pollution, insecticide resistance, rising prices for synthetic insecticides, etc. Biological control becomes an ideal strategy for mosquito control because of its effectiveness and safety in the environment.3 Bacillus thuringiensis (Bt) is a ubiquitous Gram-positive, rodshaped, and sporulating bacterium that is active to larvae of diverse insect orders.8 Since isolation of Bt in 1900, the first Bt products came in the 1930s.9 It is believed that Cry-toxininduced membrane pore formation is responsible for the toxicity against insects. When ingested by insects, Cry protoxins are hydrolyzed into active toxins by midgut proteases, which then bind to membrane receptors in the midgut cells, including aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, ATP-binding cassette (ABC) transporter, etc.10 Oligomerization of toxins and insertion of toxin oligomers into the midgut epithelial cells lead to the formation of pores and cell death.8,11 Therefore, the interaction of active toxins with toxin membrane receptors is the key step for the molecular mechanism of pore formation. However, development of resistance to Bt proteins can diminish or even eliminate the advantages of Bt products as biocontrol agents.12 Many reasons may lead to Bt resistance; for example, mutations Received: September 22, 2017 Revised: November 16, 2017 Accepted: November 22, 2017

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DOI: 10.1021/acs.jafc.7b04427 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

The supernatant was pooled and centrifuged at 30000g for 30 min at 4 °C, and the resulting pellet was resuspended in approximately 100 μL of MET buffer A per midgut. Protein Pull-Down Assay and Identification of Cry11AaBinding Proteins. To identify Cry11Aa-binding proteins in the midgut of C. quinquefasciatus larvae, purified Cry11Aa was biotinylated by EZ-Link NHS-Biotin following the instructions of the manufacturer (Thermo Fisher Scientific, Waltham, MA, U.S.A.) and biotinylated Cry11Aa (100 μL) were added to 500 μg of BBMVs of C. quinquefasciatus and incubated at 4 °C overnight with gentle rocking. The sample was centrifuged at 16000g for 1 h, and the pellet was resuspended in 1 mL of solubilization buffer [20 mM Tris−HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM MgSO4, 0.01 NaN3, 10% glycerol, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS), and 1 mM phenylmethylsulfonyl fluoride (PMSF) at pH 8.0] at 4 °C for 1 h and then centrifuged at 16000g at 4 °C for 1 h. The supernatant was then added to 50 μL of Streptavidin Agarose Beads (Thermo Fisher Scientific, Waltham, MA, U.S.A.) and incubated overnight at 4 °C. After centrifugation, the beads were washed with phosphate-buffered saline (PBS) 3 times, proteins were eluted in 100 μL of solubilization buffer, separated on SDS−PAGE, and stained by Coomassie Blue, and gels were divided and cut into seven parts for liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis according to the published method.22,28−30 Cloning, Expression, and Purification of Recombinant ATPBinding Protein. According to the sequence of C. quinquefasciatus ATP-binding protein on the National Center for Biotechnology information (NCBI) (accession number XM_001863295), polymerase chain reaction (PCR) was performed using gene-specific primers ATPF, 5′-GGA ATT CCA TAT GCA GAA GCA AGT GGC CAC CCT CCA GAC CAG GAA-3′ (the underline indicates the NdeI restriction site), and ATPR, 5-ACG CGT CGA CAC TCC GAT ATG GAT CTA CGT CGC GTT-3′ (the underline indicates the SalI restriction site), for the ATP-binding protein, with an annealing temperature of 55 °C for 1 min. The PCR product was purified using the DNA gel extraction kit (Omega), ligated into pMD18-T (following the instructions in the manual by the manufacturer), and then transformed into E. coli JM109. The sequence of the ATP-binding protein fragment in the recombinant plasmid DNA was confirmed by DNA sequencing at Sangon Biotech (Shanghai) Co., Ltd. The molecular weight and isoelectric point (pI) of the recombinant ATPbinding protein were also analyzed with ProtParam in http://www. expasy.org/vg/index/Protein. Meanwhile, structure prediction and transmembrance domain analyses were also carried out in https:// www.swissmodel.expasy.org. To express recombinant ATP-binding protein, recombinant plasmid DNA was prepared and digested with NdeI and SalI and the DNA fragment was recovered, ligated to pET-32a expression vector, and then transformed into E. coli BL21. Empty pET-32a plasmid was also transformed to E. coli BL21 for expression and purification of the control thioredoxin protein. Then, the transformants were cultivated in LB liquid medium with 0.1% ampicillin at 37 °C for 2 h and induced for protein expression with 0.3 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 16 °C for 12 h. Recombinant ATP-binding protein and recombinant thioredoxin were purified by nickel−nitrilotriacetic acid (Ni−NTA) resin affinity chromatography (TransGen Biotech, China).24 Meanwhile, the purified ATP-binding protein and the control thioredoxin protein were also biotinylated by EZ-Link NHSBiotin (Thermo Fisher Scientific, Waltham, MA, U.S.A.) according the instructions of the manufacturer. Primary rabbit polyclonal antibody against biotin and goat anti-rabbit AP-conjugated secondary antibody (Beyotime, Shanghai, China) were used in western blot and farwestern blot analyses. Western Blot. To detect expression of recombinant proteins, the biotinylated ATP-binding protein and purified Cry11Aa were separated on SDS−PAGE and transferred to nitrocellulose membrane and western blot analysis was carried out with the primary rabbit polyclonal antibody to biotin for biotinylated ATP-binding protein (1:2000) or the primary rabbit polyclonal antibody to Cry11Aa (1:2000) for Cry11Aa, followed by a goat anti-rabbit AP-conjugated

of the ABC transporter gene have been reported to be genetically linked to Bt resistance.12−17 There are also some proteins that might affect the toxicity of Cry toxins. Trypsin-like serine proteases and Dorsal [a member of nuclear factor-κB (NF-κB) transcription factors in arthropods that regulates expression of the downstream target genes in the Toll signaling pathway]18 were identified as Cry1Ab1-interacting proteins in the midgut juice of Plutella xylostella.19 Peroxidase C (POX-C) was also found to be a Cry1Ab-binding protein in Spodoptera exigua.19 However, little is known about how these proteins interact with the toxin and whether these proteins can affect the toxicity of Cry toxins.19 In this study, we focused on the proteins in the midgut of mosquitoes that can interact with Cry11Aa. We showed that an ATP-binding protein in the midgut of Culex quinquefasciatus was pulled down by recombinant Cry11Aa. Far-western blot and enzyme-linked immunosorbent assay (ELISA) analyses confirmed the interaction between Cry11Aa and the ATPbinding protein, and bioassay results showed that feeding C. quinquefasciatus larvae the recombinant ATP-binding protein significantly increased Cry11Aa toxicity. To our knowledge, this is the first report about a midgut protein that can interact with Cry toxin and enhance the toxicity of Cry toxin against mosquito larvae. Our results suggest that insect midgut proteins other than the putative Cry toxin membrane receptors can also modulate the toxicity of Cry toxins.

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MATERIALS AND METHODS

Mosquitoes, Bacterial Strains, and Plasmids. C. quinquefasciatus (Fuzhou strain) was maintained in our lab and reared in an environmentally controlled room at 28 °C and 85% relative humidity (RH) with a photoperiod of 14 h light and 10 h dark. The recombinant Bt strain pCG6 producing Cry11Aa was kindly provided by Dr. Sarjeet Gill, University of California, Riverside, Riverside, CA, U.S.A. Escherichia coli strain JM109 was used for recombinant DNA cloning, and E. coli BL21 was used for protein expression. Plasmid pMD18-T (TaKaRa, Dalian, China) was used for DNA cloning and sequencing, and pET32a is used for the expression of ATP-binding protein. Bacteria were cultivated in Luria−Bertani (LB) liquid medium at 30 °C (37 °C for E. coli) with constant rotary shaking at 230 rpm. Preparation of Cry11Aa and Its Polyclonal Antibody. Because protoxin binds to receptors of insect, such as Cry1Ab binds to cadherin,20 protoxin of Cry11Aa was prepared and used to detect the binding ability in this study. Recombinant Cry11Aa was expressed and purified from the recombinant Bt strain following the procedures described by Chen et al.21−23 Polyclonal antibodies against Cry11Aa were produced in rabbits using purified recombinant Cry11Aa as an antigen.24 Goat anti-rabbit alkaline phosphatase (AP)-conjugated secondary antibody was used for immunoblotting assays and detected by the Bcip/nbt alkaline phosphatase assay kit (Beyotime, Shanghai, China). To detect Cry11Aa protoxin, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) and western blot were carried out.24,25 Preparation of Brush Border Membrane Vesicles (BBMVs) of C. quinquefasciatus. BBMVs were prepared from the midguts of the fourth-instar C. quinquefasciatus larvae and quantified according to the published methods.22,26,27 About 1000 guts were dissected and resuspended in MET buffer A [0.3 M mannitol, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 17 mM Tris−HCl at pH 7.4, and protease inhibitor (Roche)] and then homogenized in a 1 mL Dounce homogenizer. The crude homogenate was made up to 1 mL, and an equal volume of ice-cold 24 mM MgCl2 solution was added and incubated on ice for 15 min, followed by centrifugation at 2500g for 15 min at 4 °C. The supernatant was collected and stored on ice. The precipitate was made up to 500 μL, and an equal volume of 24 mM MgCl2 was added and allowed to stand on ice for 20 min before centrifugation at 2500g for 15 min at 4 °C. B

DOI: 10.1021/acs.jafc.7b04427 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry secondary antibody (Beyotime, Shanghai, China). Antibody binding was visualized by a color reaction using the Bcip/nbt alkaline phosphatase assay kit (Beyotime, Shanghai, China).22−24 Far-Western Blot. To confirm the interaction of the ATP-binding protein with Cry11Aa, far-western blot was performed according to the method of Einarsson and Orlinick.31 The purified ATP-binding protein was separated in 10% SDS−PAGE, and protein was transferred to a nitrocellulose membrane. After washing with Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T) and blocked with 5% dry skim milk in TBS-T for 1 h, the membrane was probed with purified Cry11Aa in TBS-T containing 0.1% BSA at pH 9.0 overnight at 4 °C with a gentle rocking. After washing, the membranes were then incubated with the primary rabbit polyclonal antibody against Cry11Aa (1:2000) and goat anti-rabbit AP-conjugated secondary antibody. Antibody binding was visualized by the Bcip/nbt alkaline phosphatase assay kit (Beyotime, Shanghai, China) (1:3000). On the other side, Cry11Aa was also separated, transferred to a nitrocellulose membrane, and then probed with purified biotinylated ATP-binding protein, followed by incubation with the primary rabbit polyclonal antibody against biotin and the goat anti-rabbit AP-conjugated secondary antibody, as described above. To test the binding of Cry11Aa to C. quinquefasciatus BBMVs, BBMVs were separated on SDS−PAGE, transferred to a nitrocellulose membrane, and probed with Cry11Aa. After washing, the bound Cry fragments were detected with primary rabbit antibody to Cry11Aa, as described above. ELISA. To further confirm the interaction between Cry11Aa and ATP-binding protein, a modified microplate assay was carried out. First, 96-well plates coated with 4 μg of Cry11Aa or ATP-binding protein were incubated overnight at 4 °C and then washed with PBS 3 times. Increasing concentrations of the biotinylated ATP-binding protein (0−1280 nM), the biotinylated Cry11Aa toxin (0−1280 nM), or the control biotinylated thioredoxin protein (0−1280 nM) in 100 μL of PBST (0.1% Tween 20 and 1× PBS at pH 7.4) were then transferred to the protein-coated plates and incubated for 2 h at room temperature. The plates were washed with 100 μL of PBST 3 times, and the bound biotinylated ATP-binding protein, thioredoxin, or Cry11Aa protein was detected by incubating with streptavidin horseradish peroxidase (HRP) conjugate (1:3000) for 1.5 h. After washing, signal was detected by HRP (Beyotime, Shanghai, China) according to the instructions of the manufacturer. Bioassays. For bioassays with the purified recombinant ATPbinding protein, the third-instar C. quinquefasciatus larvae were fed purified Cry11Aa (0.85 μg/mL), Cry11Aa (0.85 μg/mL) mixed with increasing concentrations of purified recombinant ATP-binding protein, or the control thioredoxin (0, 0.36, 1.1, and 1.8 μg/mL). Survival of mosquito larvae was then recorded starting at 12 h and up to 48 h after feeding. Each treatment was replicated 3 times.

Figure 1. Purification of Cry11Aa and its binding to BBMVs. (A) SDS−PAGE analysis of purified Cry11Aa: lane M, broad protein markers; lane 1, purified Cry11Aa. (B) Binding of Cry11Aa to BBMVs by western blot analysis: lane M, broad protein markers; lane 1, BBMVs probed with purified Cry11Aa; lane 2, purified Cry11Aa.

result suggests that Cry11Aa may kill C. quinquefasciatus larvae with mechanisms similar to those of other Cry11 toxins. Identification of Cry11Aa-Binding Proteins in the Midgut of C. quinquefasciatus Larvae. It is widely believed that Cry-toxin-induced membrane pore formation is responsible for the toxicity of Cry toxins. The molecular mechanism of pore formation involves recognition and subsequent binding of the toxins to membrane receptors.35,36 Cry toxins must bind effectively to putative receptors, such as ALP, APN, cadherin, and ABCC2, to exert their toxicities. Interaction between Cry toxins and receptors should be a key step in Bti tolerance or resistance. Other than Cry toxin receptors, midgut proteins that can interact with Cry toxins may also alter the toxicity of Cry toxins. It has been reported recently that trypsin-like serine proteases and Dorsal in P. xylostella and POX-C in S. exigus can interact with Cry toxins.19 Thus, identification of Cry-binding proteins will help elucidate the insecticidal mechanism of Cry toxins.29 To identify Cry11Aa-binding proteins in the midgut of C. quinquefasciatus larvae, a biotin-based protein pull-down experiment was performed. Result showed that at least seven bands of Cry11Aa-binding proteins were detected in SDS− PAGE (bands a−g of lane 2 in Figure 2A). Analysis of these proteins by LC−MS/MS showed that there were 22 protein in these bands (Table S1 of the Supporting Information), and one of the proteins is ATP-binding domain 1 family member B (Figure 2B and Table S1 of the Supporting Information). Expression and Purification of the Recombinant ATPBinding Protein. ABC transporters comprise a large superfamily of membrane proteins.37 It has been reported that mutations in the ABCC2 gene were involved in the resistance to Cry1Ca and Cry1A toxins in a Bt commercial product.15,38 It was also found that ABCC2 is related to the mode of action of Cry1A-type proteins12 and may mediate resistance of Bt and increase susceptibility to other bacterial toxins in P. xylostella,39,40 Trichoplusia ni,39 Bombyx mori,41 Heliothis virescens,13 and even cultured cells.42 The occurrence of cadherin and ABCC2 is also thought to be necessary for the full toxicity of Cry1A proteins.13,15,42 According to the sequence of C. quinquefasciatus ATPbinding protein in NCBI (accession number XM_001863295), gene-specific DNA primers were designed, the target gene was cloned and sequenced, and the recombinant ATP-binding

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RESULTS AND DISCUSSION Purification of Cry11Aa. Bti is the first subspecies of Bt with toxicity to dipteran larvae, and Cry11Aa displays high toxicity to the larvae of Aedes and Culex mosquitoes but low toxicity against Anopheles mosquitoes.1,32−34 In this study, Cry11Aa from Bt strain pCG6 was purified. SDS−PAGE analysis showed that the apparent molecular weight of purified Cry11Aa protoxin was ∼72 kDa (lane 1 in Figure 1A), and the protein was detected by rabbit polyclonal antibody against Cry11Aa (lane 2 in Figure 1B). Binding of Cry11Aa to C. quinquefasciatus BBMVs. To test binding activity of Cry11Aa to C. quinquefasciatus BBMVs, Cry11Aa protoxin was used to probe BBMVs of C. quinquefasciatus that were separated in SDS−PAGE and transferred to a nitrocellulose membrane. The result of the ligand blot showed that purified Cry11Aa bound to C. quinquefasciatus BBMVs, because rabbit polyclonal antibody specific to Cry11Aa recognized proteins of C. quinquefasciatus BBMVs after probing with Cry11Aa (lane 1 in Figure 1B). This C

DOI: 10.1021/acs.jafc.7b04427 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Protein pull-down and identification of Cry11Aa-binding proteins. (A) SDS−PAGE analysis of Cry11Aa-binding protein: lane M, broad range protein markers; lane 1, biotinlynated Cry11Aa-bound beads; and lane 2, proteins in BBMVs pulled down by biotinlynated Cry11Aa. (B) LC− MS/MS identification of the ATP-binding protein.

protein was expressed successfully. Analysis of the C. quinquefasciatus ATP-binding protein showed that it was not similar to ABCC2 transporters. Bioinformatic analysis showed that the open reading frame (ORF) of this ATP-binding protein consists of 924 nucleotides, which encode a protein of 307 amino acids with a calculated molecular weight of 34.72 kDa and pI value of 5.47. The C. quinquefasciatus ATP-binding protein is most similar to proteins from Culex tarsalis (96% identity, accession number JAV22961), A. aegypti (84% identity, accession number XP_001661906), and A. albopictus (83% identity, accession number XP_019543508). Secondary structure prediction of the C. quinquefasciatus ATP-binding protein showed that it contains 56.35% α-helix, 7.17% β-sheet, and 20.52% random coil and does not contain a transmembrance domain. Thus, the ATP-binding protein is not similar to ABC transporters. Because the ABC transporter is involved in the resistance to Cry toxins, we also like to know the function of the ATP-binding protein in Bt toxicity/ tolerance. Binding of the Recombinant ATP-Binding Protein to Cry11Aa. To test binding of the ATP-binding protein to Cry11Aa, the recombinant ATP-binding protein was expressed and purified as analyzed by SDS−PAGE (arrow in Figure 3A).

3B). When the purified ATP-binding protein in the membrane was probed with Cry11Aa and detected with rabbit polyclonal antibody specific to Cry11Aa, the 35 kDa biotinylated ATPbinding protein was observed (arrow in Figure 3C). Similarly, 72 kDa Cry11Aa in the membrane was also detected by rabbit polyclonal antibody specific for biotin after probing with the biotinylated ATP-binding protein (arrowhead in Figure 3D). To confirm binding of the ATP-binding protein to Cry11Aa, plate ELISAs were also performed. The results showed that more biotinylated ATP-binding protein bound to immobilized Cry11Aa with increasing concentrations of the ATP-binding protein and the binding was not saturated at 1250 nM ATPbinding protein (Figure 4A). Similarly, more biotinylated Cry11Aa bound to immobilized ATP-binding protein when Cry11Aa concentrations were increased, and the binding was saturated after 320 nM Cry11Aa (Figure 4B). A neglected amount of the control thioredoxin protein bound to either immobilized ATP-binding protein or Cry11Aa (Figure 4). Together, these results indicate that the ATP-binding protein can interact with Cry11Aa. Increased Insecticidal Activity of Cry11Aa by the ATPBinding Protein. To test whether the interaction of the ATPbinding protein with Cry11Aa may affect the toxicity of Cry11Aa, bioassays were carried out with a fixed concentration of Cry11Aa in the presence of increasing concentrations of the recombinant ATP-binding protein or a control thioredoxin protein. Bioassay results showed that feeding mosquito larvae Cry11Aa mixed with recombinant thioredoxin did not affect larvae survival compared to feeding Cry11Aa alone (0 μg/mL protein) at 12, 24, and 48 h post-feeding (Figure 5A). However, the survival of mosquito larvae significantly decreased when larvae were fed Cry11Aa mixed with the recombinant ATP-binding protein compared to feeding Cry11Aa alone at the three time points post-feeding depending upon the concentrations of the ATP-binding protein (Figure 5B). All of the tested mosquito larvae survived after feeding on the ATP-binding protein at the different concentrations, which might deduce that the binding completion might relate to the toxicity reduction of Cry11Aa. These results indicate that the interaction between the ATP-binidng protein and Cry11Aa enhances the toxicity of Cry11Aa against C. quinquefasciatus larvae. In this study, we identified a Cry11Aa-binding protein from the midgut of C. quinquefasciatus larvae and confirmed the interaction between the ATP-binding protein and Cry11Aa. Importantly, we showed that the interaction of the ATPbinding protein with Cry11Aa enhanced the toxicity of Cry toxins against mosquito larvae. This is the first report about Cry-binding-secreted proteins from the midgut that can enhance the toxicity of Cry toxins. Our results suggest that

Figure 3. Western and far-western blot analyses of the ATP-binding protein and Cry11Aa. (A) SDS−PAGE analysis of the purified recombinant ATP-binding protein (ABP). (B) Western blot analysis of the biotinylated ABP. (C) Far-western blot analysis of the purified ABP probed with Cry11Aa. (D) Far-western blot analysis of Cry11Aa probed with biotinylated ABP. Arrows indicate ABP, while an arrowhead indicates Cry11Aa.

Binding of the biotinylated ATP-binding protein to Cry11Aa was first tested by far-western blot analysis. We showed that purified Cry11Aa was recognized by the primary rabbit polyclonal antibody to Cry11Aa (lane 2 in Figure 1B) and the biotinylated ATP-binding protein was recognized by the primary rabbit polyclonal antibody to biotin (arrow in Figure D

DOI: 10.1021/acs.jafc.7b04427 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. Interaction between Cry11Aa and the ATP-binding protein. The 96-well plates were coated with a fixed amount of (A) Cry11Aa or (B) recombinant ATP-binding protein (ABP), and increasing concentrations of the biotinylated ABP, biotinylated Cry11Aa, or thioredoxin (control protein) were then added to each well. Binding of (A) biotinylated ABP to Cry11Aa or (B) biotinylated Cry11Aa to ABP was determined by plate ELISAs, as described in the Materials and Methods. Each point represents the mean of three independent measurements ± standard error of the mean (SEM), and the lines represent the nonlinear regression for on-site binding.

Figure 5. Bioassay of Cry11Aa in the presence of the ATP-binding protein or thioredoxin. C. quinquefasciatus larvae were treated with purified Cry11Aa in the presence of increasing concentrations of (A) control recombinant thioredoxin or (B) recombinant ATP-binding protein, and the survival of mosquito larvae was recorded from 12 to 48 h after treatment.

for Scientific and Technological Innovation of Fujian Agriculture and Forestry University (Grants KF2015063-65, CXZX2017136, and CXZX2017306).

there may be many other midgut proteins that can interact with Bt toxins and alter the toxicity of Bt.

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ASSOCIATED CONTENT

Notes

S Supporting Information *

The authors declare no competing financial interest.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04427. All checked Cry11Aa-binding proteins identified by LC− MS (PDF)

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ACKNOWLEDGMENTS The authors are thankful to Dr. Sarjeet Gill (University of California, Riverside, Riverside, CA, U.S.A.) for providing the recombinant Bt strain.

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AUTHOR INFORMATION

ABBREVIATIONS USED Bt, Bacillus thuringiensis; BBMV, brush border membrane vesicle; APN, aminopeptidase N; ALP, alkaline phosphatase; ABC, ATP-binding cassette; LB, Luria−Bertani; PCR, polymerase chain reaction; POX-C, peroxidase C; IPTG, isopropyl-β-Dthiogalactoside; SDS−PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; LC−MS/MS, liquid chromatography−tandem mass spectrometry; NCBI, National Center of Biotechnology Information

Corresponding Authors

*Telephone: +1-816-235-6379. E-mail: [email protected]. *Telephone/Fax: +86-591-8378-9259. E-mail: guanxfafu@126. com. ORCID

Xiong Guan: 0000-0003-3505-7464 Funding

This work was supported by National Key R&D Program of China (2017YFD0200400 and 2011AA10A203), the Fujian− Taiwan Joint Center for Ecological Control of Crop Pests [Minjiaoke (2013)51], the National Natural Science Foundation of China (NSFC, Grant 31301724), the Leading Talents of Fujian Province College (K8012012a), and the Special Fund

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REFERENCES

(1) Ben-Dov, E. Bacillus thuringiensis subsp. israelensis and its Dipteran-specific toxins. Toxins 2014, 6, 1222−1243.

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Journal of Agricultural and Food Chemistry

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DOI: 10.1021/acs.jafc.7b04427 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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