Expression of Vip3 proteins and engineering of DIP mutants
All Vip3 proteins contained an Nt 6x-His tag. Vip3Aa16 (ac. no. AAW65132) was expressed in recombinant Escherichia coli BL21 cells34 following the protocol described elsewhere35. Vip3Ca2 (ac. no. AEE98106)22, Vip3Af1 (ac. no. CAI43275) and mutant Vip3Af E168A (kindly provided by BASF Belgium Coordination Center – Innovation Center Ghent) were expressed in recombinant E. coli WK6 cells following a protocol described previously36.
For constructing the Vip3 disabled proteins, the mutation S164C L166C was selected based on the results reported by Jerga et al.21 where a Vip3Aa DIP variant was generated by the introduction of two cysteines in enough proximity to make a S–S bond and prevent Domain I to unfold, a critical step in the activation of the Vip3 protoxins11,12. The mutation E168A was selected based on previous studies showing that Vip3Af E168A loses toxicity but retains the tetrameric structure and we hypothesized that could also behave as a DIP mutant14,16. The DIP proteins Vip3Aa S164C L166C, Vip3Aa E168A and Vip3Ca S164C L166C were generated for this work by SDM following the protocol described elsewhere37, using the WT clones as templates. Primers and annealing temperatures used for SDM Polymerase Chain Reaction (PCR) are collected in Supplementary Table S2. The resulting mutant clones were expressed and lysed following the same conditions used for their WT homologues.
Purification of Vip3 proteins
For the in vitro binding assays, proteins were purified by metal-chelate affinity chromatography using His-trap FF crude (GE Healthcare) 1 ml columns35. Briefly, the clarified lysate was loaded onto the column previously equilibrated with 50 mM phosphate buffer containing 300 mM NaCl and 10 mM imidazole. Then, the column was washed with the same buffer and an increased imidazole concentration (45 mM), and the His-tagged Vip3 protein bound to the column was finally eluted rising imidazole concentration to 250 mM in the same buffer. Purified proteins were dialyzed overnight against 20 mM Tris–HCl, 150 mM NaCl (pH 8.6) buffer, and after dialysis, they were clarified by centrifugation (16,100×g, 4 °C, 10 min), quantified by the Bradford method38 using bovine serum albumin (BSA) as standard, aliquoted and stored at − 20 °C. The purity of the protein preparations was checked by SDS-PAGE (Supplementary Fig. S2). Just before use, purified Vip3 proteins used as competitors were trypsin-treated (1% w/w) with trypsin from bovine pancreas (SIGMA T8003, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h, clarified by centrifugation (16,100×g for 10 min at 4 °C), and quantified by Bradford.
In the in vivo competition assays, we observed that His-trap purified proteins rendered less reproducible results compared to the isoelectric point precipitation (IPP) purified proteins, most probably due to a higher stability of the latter on the surface of the diet for 10 days at 25 °C. For this reason, proteins meant to be used for in vivo competition assays were purified by IPP35. Briefly, the clarified lysate was lowered at pH 5.6 for Vip3Aa and Vip3Af (WT and mutant proteins) and at pH 5.9 for Vip3Ca (WT and mutant protein) with 0.1 M acetic acid. Then, the lysate with the adjusted pH was aliquoted and the precipitation pellet was recovered by centrifugation (16,100×g, 4 °C, 10 min) and stored at – 20 °C. Before being used in bioassays, the pellet aliquots were dissolved in 20 mM Tris–HCl, 150 mM NaCl (pH 8.6) buffer, and clarified by centrifugation (16,100×g, 4 °C, 10 min). Protein purity was checked by SDS-PAGE (Supplementary Fig. S3) and the protein concentration was calculated densitometrically using BSA as standard and the TotalLab 1D v 13.01 software.
S. littoralis brush border membrane vesicles (BBMV) preparation
Last instar larvae of S. littoralis were dissected and midguts were kept at − 80 °C. BBMV were prepared from the midguts by the differential magnesium precipitation method39, flash frozen in liquid nitrogen, and stored at − 80 °C until use. The protein concentration in the BBMV preparations was determined by Bradford.
Vip3Aa 125I radiolabeling
Radiolabeling of Vip3Aa protoxin was performed by the chloramine T method40 following the protocol described by Chakroun and Ferré7 with some modifications. His-trap purified Vip3Aa (25 µg) was mixed with 0.3 mCi of [125I]-NaI and 1/3 (vol/vol) 18 mM Chloramine T. The excess of free 125I was separated from the labeled protein using a PD10 desalting column (GE Healthcare). The purity of the 125I labelled protein was checked by analyzing the eluted fractions by SDS-PAGE with further exposure of the dry gel to an X-ray film (Supplementary Fig. S4). The specific activity of the labeled 125I-Vip3Aa was 5.7 µCi/µg.
In vitro binding assays with 125I -Vip3Aa and S. littoralis BBMV
Before use, labelled 125I-Vip3Aa protein was trypsin treated (1% w/w, 37 °C, 1 h) and stored at 4 °C. BBMV were thawed on ice, centrifuged (10 min at 16,000×g and 4 °C) and resuspended in binding buffer (20 mM Tris–HCl, 1 mM MnCl2, 0.1% BSA, pH 7.4).
For all the assays, trypsin-treated 125I-Vip3Aa was incubated with BBMV for 1 h at room temperature in a 0.1 ml final volume of binding buffer. The reaction was stopped by centrifuging the tubes at 16,000×g for 10 min at 4 °C, and the pellet was washed once with 500 µl of cold binding buffer7. The radioactivity retained in the pellet was measured in a model 2480 WIZARD2 gamma counter. A binding assay with a fixed amount of 125I-Vip3Aa (0.2 nM), increasing concentrations of BBMV and an excess of unlabelled competitor (1000-fold His-trap purified, trypsin-treated, Vip3Aa) was performed to determine the appropriate amount of BBMV to use in the binding competition assays and estimate nonspecific binding (Supplementary Fig. S1). Competition assays were performed by mixing a fixed amount of 125I-Vip3Aa (0.2 nM) with increasing concentrations of unlabelled competitor (His-trap purified, trypsin-treated, Vip3 proteins) and a fixed amount of BBMV (0.05 mg/ml). At least three replicates were performed for each binding or competition assay. Graphical representations were performed with GraphPad Prism version 5. Equilibrium dissociation constants (Kd) and the concentration of binding sites (Rt) were estimated using the LIGAND software41.
Insect rearing and bioassays
Surface contamination bioassays were performed with a laboratory population of S. littoralis maintained on semi-synthetic diet42 in a rearing chamber at 25 ± 2 °C, 70 ± 5% RH and 16:8 h L:D.
To calculate the LC50 values of WT proteins, increasing doses of proteins purified by isoelectric point precipitation (IPP) dissolved in Tris buffer (20 mM, 150 mM NaCl, pH 8.6) were prepared. Protein preparations were dispensed (50 µl) over the surface of the assay wells (2 cm2 of diameter) filled with semi-synthetic diet, sixteen wells were used for each protein dose. After the diet surface was dry, one neonate was gently placed into each well and the plates were sealed. Trays were maintained in a climatic chamber at the same conditions used for colony rearing and mortality was recorded after 7 and 10 days of incubation. At least three replicates were performed for each protein, and the LC50 values and FL95% were calculated using PoloPlus software.
For in vivo competition assays, protein:competitor ratios of 1:0, 1:10, 1:100 and 1:1000 were prepared in Tris buffer (20 mM, 150 mM NaCl, pH 8.6). Controls consisted of just buffer (labeled as 0) and just competitor (labeled as 0:1000, corresponding to the maximum amount of competitor used in the assay). Constant concentrations of IPP-purified Vip3Aa (13 ng/cm2) and Vip3Af (30 ng/cm2), which produced 60–80% of mortality, were mixed with the corresponding amount of IPP-purified competitor to prepare the different ratios. Bioassays were performed following the same protocol described previously, and at least three biological replicates were performed for each in vivo competition protein combination. Graphical representations were performed with GraphPad Prism version 5, and for assessing the statistical significance of mortality differences between 1:0 and the rest of protein:competitor ratios, a One way Anova with post-hoc Bonferroni test was performed using the same software.
