APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea

Lepidopteran midgut aminopeptidases N (APNs) are phylogenetically divided into eight clusters, designated as APN1–8. Although APN1 has been implicated as one of the receptors for Cry1Ac in several species, its potential role in the mode of action of Cry2Ab has not been functionally determined so far. To test whether APN1 also acts as one of the receptors for Cry1Ac in Helicoverpa zea and even for Cry2Ab in this species, we conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by RNAi (RNA interference) silencing of the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA (dsRNA). Heterologous expression of HzAPN1 significantly increased the susceptibility of the three cell lines to Cry1Ac, but had no effects on their susceptibility to Cry2Ab. Knocking down of the endogenous APN1 made the three cell lines resistant to Cry1Ac, but didn’t change cell lines susceptibility to Cry2Ab. The findings from this study demonstrate that HzAPN1 is a functional receptor of Cry1Ac, but not Cry2Ab.

their involvement in Cry1Ac resistance has not been documented. APN6 seems not to be a receptor for Cry1Ac and Cry2Ab because it can not bind to Cry1Ac in H. armigera 19 and is not involved in Cry1Ac and Cry2Ab resistance in Trichoplusia ni 14,20 . In stark contrast, various studies with APN1 from Manduca sexta 21 , L. dispar 22,23 , P. xylostella 24 , H. armigera 15,25 , and T. ni 20 consistently demonstrate that it is one of the midgut receptors for Cry1Ac.
So far, the only study examined APN's role in the mode of action of Cry2Ab shows that APN5 can bind to Cry2Ab in P. xylostella 26 . Whether APN1 also serves as one of the receptors for Cry2Ab still remains unclear. Presence of cross-resistance between Cry1Ac and Cry2Ab [27][28][29] and detection of a Cry1Ac-and Cry2Ab-binding protein with a similar size (120 ~ 130 kDa) of APN1 in the midgut and fat body cell lines of H. zea by ligand blot suggest a possibility of APN1 as a shared receptor for Cry2Ab and Cry1Ac (Wei & Li, unpublished manuscript). To test this hypothesis, we conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by knocking down the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA (dsRNA). Heterologous expression of HzAPN1 significantly enhanced the susceptibility of the three cell lines to Cry1Ac, but not to Cry2Ab. Knocking down of the endogenous APN1 made the three cell lines more tolerant to Cry1Ac, but didn't change their susceptibility to Cry2Ab. The data demonstrate that APN1 is a receptor for Cry1Ac, but not for Cry2Ab.

Results
Binding profiles of Cry1Ac and Cry2Ab in H. zea larval midgut BBMV. As  Impact of heterologous expression of HzAPN1 on cytotoxicities of Cry1Ac and Cry2Ab. Western blot showed that Sf9 cells and H. zea midgut and fat body cells transfected with pAc-HzAPN1 produced significantly more APN1 proteins than the three cell lines transfected with the empty vector pAc (control cells) ( Fig. 2; P midgut = 0.035, P fat body = 0.004 and P Sf9 = 0.048). More precisely, the protein level of APN1 in the pAc-HzAPN1 transfected midgut, fat body and Sf9 cells was 1.83, 1.55 and 1.97 times more than that in the corresponding pAc-transfected control cells.
When the above pAc-and pAc-HzAPN1-transfected cells were exposed to 15 μ g/ml of the activated Cry1Ac, mortality increased from none (− 2.64%) for pAc-transfected Sf9 cells to 48.05% for pAc-HzAPN1 transfected Sf9 cells (Table 1). Consistent with the higher APN1 increase in the pAc-HzAPN1 transfected midgut cells than in the pAc-HzAPN1 transfected fat body cells (Fig. 1), the mortality of the former increased by 55.95%, whereas it was 20.90% for the latter. When the concentration of activated Cry1Ac was doubled, mortality was increased significantly (for at least two to three times) from 24.19 (pAc control) to 76.17% (pAc-HzAPN1) for midgut cells, 23.77 to 51.57% for fat body cells and 10.61 to 44.35% for Sf9 cells, respectively (Table 1). When the pAc and pAc-HzAPN1 transfected cells were treated with 2.5 or 3.75 μ g/ml of the activated Cry2Ab, no significant mortality enhancement by heterologous expression of HzAPN1 was observed regardless of cell line and Cry2Ab concentration ( Table 1).
Effect of knocking down APN1 on cytotoxicities of Cry1Ac and Cry2Ab. Western blot detected 7.87-fold reduction in the protein level of APN1 in H. zea midgut cells transfected with 50 nM HzAPN1 dsRNA, compared with H. zea midgut cells transfected with 50 nM DsRed dsRNA (control cells; P midgut < 0.0001) (Fig. 3). Likewise, transfection of 50 nM HzAPN1 dsRNA also resulted in 2.47-(P fat body = 0.0098) and 3.74-fold (P Sf9 < 0.0001) decrease in the protein level of the endogenous APN1 in H. zea fat body cells and Sf9 cells, respectively (Fig. 3). When the above HzAPN1 dsRNA-transfected H. zea midgut cells were exposed to 150 μ g/ml of the activated Cry1Ac, a 33.79% decrease in mortality was exhibited when compared with that of the DsRed dsRNA-transfected H. zea midgut cells (Table 2). Due to the lower knocking down activities of the endogenous APN1 in the other two cell lines (Fig. 3), HzAPN1 dsRNA only produced a 19.29% and a 13.12% reduction in mortality for H. zea fat body cells and Sf9 cells, respectively ( Table 2). In contrast, no significant mortality reduction by knocking down of the endogenous APN1 was seen in the three insect cell lines when they were treated with 30 μ g/ml of the activated Cry2Ab (Table 2).

Discussion
Both Cry1Ac and Cry2Ab are expected to have two or more receptors in the larval midguts of Lepidopteran insects (Wei & Li, unpublished manuscript). Documentation of cross-resistance between the two toxins implies that they may share one common receptor [27][28][29] , although cross-resistance can occur through receptors-independent mechanisms. Recognition of one protein band with the same size of APN1 (120 ~ 130 kDa) by both Cry1Ac and Cry2Ab antibodies in the midgut and fat body cell lines of H. zea (Wei & Li, unpublished manuscript) and by  Cry2Aa antibody in Spodoptera littoralis BBMV 31 suggest that the potential common receptor could be APN1. This present study was conducted to test whether this hypothesis is true or not. By ligand blot and Western blot, we confirmed that H. zea larval midgut BBMV, like H. zea midgut and fat body cell lines (Wei & Li, unpublished manuscript), also had a 130 kDa protein band that was recognized by Cry1Ac, Cry2Ab, and APN1 antibodies (Fig. 1). Heterlogous expression of HzAPN1 significantly increased the susceptibilities of Sf9, H. zea midgut and fat body cell lines to activated Cry1Ac ( Fig. 2 and Table 1), whereas RNAi knocking down of the endogenous APN1 made the three cell lines significantly less susceptible to activated Cry1Ac ( Fig. 3 and Table 2). In contrast, neither heterologous expression of HzAPN1 nor RNAi knocking down of the endogenous APN1 affected the susceptibilities of the three cell lines to activated Cry2Ab (Tables 1 and 2). The data suggest that the 130 kDa band recognized by Cry1Ac antibody is HzAPN1, but the 130 kDa band recognized by Cry2Ab is an unknown protein. Moreover, the data directly demonstrate that HzAPN1 is one of the receptors for Cry1Ac, but not for Cry2Ab in H. zea.
While APN1 has been implicated as one of the receptors for Cry1Ac in a number of species 15,21,23 , this is the first report that functionally characterized APN1 as a receptor of Cry1Ac in H. zea. This is consistent with the   32 , the closest orthologue of HzAPN1, acted as a receptor of Cry1Ac in Sf21 cells. To our knowledge, the current study is also the first report that experimentally excluded APN1 as the basis of cross-resistance between the two toxins-the common receptor of Cry1Ac and Cry2Ab. However, this study cannot be extrapolated to rule out the possibility of other APNs as one of the receptors for Cry2Ab. First, the size of the protein band (120 ~ 130 kDa) recognized by Cry2Ab antibody not only matches with the size of APN1, but also falls into the size range of other APNs. In addition, APN5 from P. xylostella (PxAPN5) was reported to bind to Cry2Ab 26 . Further functional analyses of HzAPN5 and other APNs from H. zea are needed to resolve this issue. The fact that RNAi knocking down of the endogenous APN1 protected not only midgut cells but also fat body and ovarian (Sf9) cells from the toxic effects of Cry1Ac (Fig. 3 and Table 2) suggests that APN1 may be expressed in both midgut and non-gut hemocoelic tissues at least in some species. Consistent with this notion, APN1 has been shown to be expressed in midgut, malpighian tubule, salivary gland and fat body in T. ni 33 , fat body, malpighian tubule and salivary gland in Achaea janata 34 , midgut, fat body, malpighian tubule, epidermis and hemolymph in S. exigua 35 , midgut, fat body, Malpighian tubule and carcass in Epiphyas postvittana 36 , and midgut and Malpighian tubule in B. mori 37 . The toxic effects of Cry1Ac and Cry2Ab on in vitro cultured fat body and ovarian (Sf9) cells observed in this study and Wei et al. (Wei & Li, unpublished manuscript) as well as the effects of intrahemocoelic injections of several Cry1 toxins on the growth and survival of Lymantria dispar and Neobellieria bullata larvae 38 and of A. janata larvae 34 further strengthen the notion of APNs expression in the above non-gut hemocoelic tissues. Apparently, these receptors-expressing non-gut hemocoelic tissues can serve as additional in vivo target tissues of Cry toxins, as long as these toxins can cross the insect midgut epithelium and reach the haemolymph when fed orally. Detection of small amounts of orally ingested Cry toxins in the haemolymph of Lygus Hesperus 39 proves that Cry toxins can naturally cross the insect gut into the haemolymph at least in some species, but probably at a very low speed. Novel protein conjugation/fusion technology capable of speeding up the crossing of the haemocoelic-active Cry toxins across the insect gut into haemolymph is needed to further enhance the efficacy of Cry toxins by virtue of targeting both midgut and non-gut hemocoelic tissues. Ligand blot detection of CryAc and Cry2Ab binding proteins in H. zea larval midgut BBMV. Brush border membrane vesicles (BBMV) for ligand blot and Western blot (see below) were prepared from the 5 th instar larvae midguts of the susceptible strain of H. zea using a differential centrifugation method 42,43 . When dissected out, midguts were open to remove peritrophic membranes and gut contents, cleaned in ice-cold 0.7% NaCl solution, placed on filter paper for a few seconds to remove excessive water, weighed and stored at − 80 °C. The frozen midguts taken out from the freezer were homogenized in nine-fold volume (w/v) of ice-cold buffer A (300 mM mannitol, 5 mM EGTA, 17 mM Tris-HCl, 1 mM PMSF), incubated with one volume of 24 mM MgCl 2 on ice for 15 min, and centrifuged at 2500 × g and 4 °C for 15 min. The supernatant was centrifuged at 30,000 × g and 4 °C for 30 min. The resulting pellet was suspended in buffer B (150 mM Mannitol, 2.5 mM EGTA, 8.5 mM Tris-HCl, 1 mM PMSF), left on ice up to 4 h, and then centrifuged at 30,000 g and 4 °C for 15 min. The final pellet was suspended in buffer C (150 mM NaCl, 5 mM EGTA, 1 mM PMSF, 20 mM Tris-HCl, 1% CHAPS) and used as the BBMV preparation. The protein concentrations of the BBMV preparations were determined by the Bradford protein assay, using bovine serum albumin (BSA) as a standard (Bradford, 1976). When conducted ligand blot, 8 μ g protein of the larval midgut BBMV preparation was separated on a 10% SDS-PAGE gel and electroblotted to a polyvinylidene difluoride (PVDF) membrane (Thermo scientific) in the transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The follow-up procedures for detection of Cry1Ac or Cry2Ab binding proteins using the anti-Cry1Ac antibody or anti-Cry2Ab antibody were the same as described in Wei et al. (Wei & Li, unpublished manuscript).

Insects and insect cell lines.
Western blot detection of APN1 in larval midgut BBMV and cell line protein extracts. The larval midgut BBMV was prepared as above and the protein extracts of the three insect cell lines transfected with the control plasmid pAc, pAc-HzAPN1, DsRed double strand RNA (dsRNA), or HzAPN1 dsRNA (see preparation and transfection of pAc-HzAPN1 plasmid and dsRNA below) were prepared as described by Wei  of the cells transfected with DsRed dsRNA (control) and HzAPN1 dsRNA were 150 μ g/ml for activated Cry1Ac and 30 μ g/ml for activated Cry2Ab, respectively. Cell mortality was determined by replacing 35 μ L medium from each well with 35 μ L of 0.4% trypan blue (AMRESCO ® , BioExpress, Ohio, USA) and counting the numbers of stained (dead cells) and unstained (live cells) in two 400x fields of view from the central parts of each well under an inverted microscope (VistaVision, VWR) after 4.5 h for Cry1Ac or 5.5 hours for Cry2Ab (Wei & Li, unpublished manuscript). Each plasmid or dsRNA was independently transfected into each cell three times and each independent transfection was bioassayed three times with control or a given concentration of Cry1Ac or Cry2Ab (3 × 3 = 9 replicates).

Statistical Analysis
Cell mortality was calculated by dividing the number of dead cells (the number of cells before toxin treatment -the number of unstained cells after treatment) by the number of cells before treatment and corrected with the Abbott formula 44 . Significant differences in mortality between the control plasmid pAc (or the DsRed control dsRNA) and pAc-HzAPN1 (or HzAPN1 dsRNA) were compared using Student t-test with α = 0.05 (JMP 8; SAS Institue, Inc.). Mortality values of different treatments were arcsine transformed before analysis.
The band intensities of the target protein APN1 and the reference protein β -actin on all the Western blot pictures of the protein extracts from the three cell lines transfected with pAc, pAc-HzAPN1, DsRed dsRNA, or HzAPN1 dsRNA were quantified by densitometry using Image J software (NIH, v1.46). The relative expression level of APN1 in each control or treatment cells was calculated by dividing the band intensity of APN1 by the band intensity of β -actin. Significant differences in the relative expression of APN1 between the control plasmid pAc (or the DsRed control dsRNA) and pAc-HzAPN1 (or HzAPN1 dsRNA) were evaluated by Student's t-test (JMP 8; SAS Institue, Inc.).