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In this work, we characterized 2 novel insecticidal proteins; Vip3Ab1 and Vip3Bc1. These proteins display unique insecticidal spectra and have differential rates of processing by lepidopteran digestive enzymes. Furthermore, we have found that both proteins exist as tetramers in their native state before and after proteolysis. In addition, we expressed truncated forms and protein chimeras to gain a deeper understanding of toxin specificity and stability. Our study confirms a role for the C-terminal 65kDa domain in directing insect specificity. Importantly, these data also indicate a specific interaction between the 20kDa amino terminus and 65kDa carboxy terminus, after proteolytic processing. We demonstrate the C-terminal 65kDa to be labile in native proteolytic conditions in absence of the 20 kDa N-terminus. Thus, the 20kDa fragment functions to provide stability to the C-terminal domain, which is necessary for lethal toxicity against lepidopteran insects.
Bacillus thuringiensis (Bt) is the bacterial source of insecticidal proteins that have been expressed in genetically modified crops to confer resistance to pest feeding damage and crop loss. Many insecticidal proteins are produced as crystalline inclusion bodies during the late log-growth to sporulation phase of bacterial growth. Insecticidal crystals are then solubilized in the digestive tract of insects where they are activated by midgut proteases to form active toxins, which disrupt membrane integrity and cause midgut epithelial cell lysis through specific interaction with cellular receptors1, 2. Another class of Bt toxins are produced during the vegetative growth phase and are termed Vegetative insecticidal proteins (Vip’s,3). The Vip proteins are composed of 4 subgroups; Vip1, Vip2, Vip3 and Vip4. Vips 1 and 2 are frequently active against coleopteran species of insects and function as binary insecticidal proteins utilizing ADP-ribosyltransferase activity to exert toxic effects on target insect cells2, 4–6. Vip3 proteins have activity against a broad spectrum of economically important lepidopteran insects such as fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), corn earworm (Helicoverpa zea), and marginal activity against European corn borer (Ostrinia nubilalis)3, 7–9. The Vip4 subgroup is composed of only one member, Vip4Aa1, with no known insecticidal activity5.
Because of their broad spectrum of insecticidal activity, Vip3 proteins have been used for the development of transgenic crops such as event COT102 cotton and event MIR162 corn10. Thus, the Vip3 subfamily is amenable to transgenic plant protection; however, much less is understood about Vip3 mode of action. The proposed sequence of biochemical events leading to toxic effect is similar to those described for classic crystalline Bt three domain toxins2. In the current proposed mode of action model, Vip3 proteins are first ingested and then activated by proteolytic removal of a ~20 kDa N-terminal pro-domain. Next, the ~65kDa active toxin “core” is proposed to bind specific receptors in the insect midgut epithelium resulting in cell lysis and loss of membrane integrity11–14. However, there is little direct biochemical evidence to substantiate this sequence of events and the majority of research has been performed on a single subclass of Vip3A proteins that share greater than 95% amino acid sequence identity. A more detailed understanding of the Vip3 mechanism of action, activation, and specificity is needed to support further development of new Vip3 insecticidal proteins as insect resistance traits.
In this work, we have functionally characterized ~20kDa and ~65kDa Vip3 domain interactions using two new members of the Vip3 family, Vip3Ab1 and Vip3Bc1. These insecticidal proteins demonstrated clear differences in proteolytic processing that did not correlate with activity against susceptible insects. In addition, we observed that both proteins exist as tetramers prior to stepwise enzymatic processing by midgut enzymes. Interestingly, Vip3Ab1 and Vip3Bc1 proteins persist as tetramers in solution after enzymatic processing, indicating a direct and sustained interaction between the products of partial proteolysis. We have also expressed and purified the C-terminal portions of Vip3Ab1 and Vip3Bc1, often referred to as the toxic “core”5, and observed no insecticidal activity for either protein. Lastly, we have synthesized chimeric Vip3 proteins to demonstrate that the C-terminal ~65kDa domain is responsible for toxin specificity and that the ~20 kDa N-terminal maintains proteolytic stability and is required for toxicity. To our knowledge, this is the first demonstration that Vip3 insecticidal proteins require interaction between amino- and carboxy- termini for toxicity. Despite highly conserved sequence identity of the amino terminal regions, our data indicate that interactions of the amino terminal regions with the carboxy terminus promote oligomerization and provide proteolytic stability required for lethal toxicity. Thus, we have demonstrated that the Vip3 family of proteins undergo a unique processing and activation pathway that is different than the 3 domain Cry family of Bt proteins. These new data shed new light on Vip3 domain functionality, indicating that Vip3 proteins may have additional potential for the control of commercially important lepidopteran pests.
An amino acid sequence alignment of Vip3Ab1 and Vip3Bc1 is illustrated in Fig. 1. By conventional terminology, members of the Vip3 class are at least 45% identical and a shared secondary ranking (A, B, etc.) indicates that two Bt proteins are at least 78% identical across the length of the protein15, 16. Vip3Bc1 has 61% amino acid identity to Vip3Ab1. The Vip3Bc1 N-terminal sequence is unique as the N-terminal methionine is 8 amino acids upstream of the more conserved sequence, MANMNNTKLN, found in Vip3Ab1 and other Vip3 family members3, 17. Vip3Bc1 also has a short insertion of 472KEKSCEEDSCEDE484, which is similar to a longer repeat of charged amino acids found in Vip3Ba1 (472KEDCCEEDCCEEDCCEEDCCEE493)18. In total, Vip3Bc1 is comprised of an additional 26 amino acids relative to Vip3Ab1 and will used as the reference sequence to for amino acid location of both proteins. Both proteins expressed with only the first methionine removed and observed molecular weight consistent with the predicted mass of ~85kDa (Fig. 2). Vip3Ab1 contains a canonical serine proteinase cleavage motif at 205 KVKK↓DSSP, whereas Vip3Bc1 contains 3 substitutions at this site resulting in 205 KSYQ↓DNVT. Both genes expressed soluble proteins in Pseudomonas fluorescens and had insecticidal activity against Pseudoplusia includens (Table 1). Vip3Ab1 had lethal activity on H. zea and S. frugiperda whereas Vip3Bc1 had lethal activity against O. nubilalis.
To investigate the role of processing and regions of diversity from Vip3Ab1 and Vip3Bc1, chimeric proteins Vip3_AB (N-terminal domain of Vip3Ab1 and C-terminal domain of Vip3Bc1) and Vip3_BA (N-terminal domain of Vip3Bc1 and C-terminal domain of Vip3Ab1) were expressed and purified from P. fluorescens. Glutamic acid at position #224 (224ELAKS) was chosen as the site to generate chimeras because this was within a short sequence conserved between Vip3Ab1 and Vip3Bc1 from amino acids 221–233 (based on Vip3Bc1 numbering, Fig. 1). Sequence exchange at this site avoided disruption of upstream processing sites, which made these proteins ideal for investigation of domain function and processing. Both toxin chimeras expressed as full length proteins with the N-terminal methionine removed. However, Vip3_BA was also present as a partially processed protein with the N-terminus at 190NEKFD (Fig. 2). Neither chimera produced lethal or morbid effects against the 4 insects utilized in this study. However, Vip3_BA-treated H. zea, P. includens, and S. frugiperda were smaller in size and showed strong inhibition of growth relative to buffer-treated insects (Fig. 3) whereas Vip3_AB showed no toxic effects on any of the tested insects.
We analyzed the time course of H. zea and P. includens midgut proteolytic processing of Vip3Ab1 and Vip3Bc1 proteins by SDS-PAGE (Fig. 4, top panel). All digestion experiments were performed at pH 10.0 as this pH is similar to the alkaline midgut environment of lepidoptera. In this condition, we observed the formation of an additional Vip3Ab1 band at ~110kDa that was not apparent in gels using protein in pH 8.0 buffer. This band and the full length ~85kDa Vip3Ab1 behaved similar during the timecourse of digestion experiments. Both Vip3Ab1 and Vip3Bc1 were processed to ~65kDa and ~20kDa products. However, Vip3Bc1 was processed at a much slower rate than Vip3Ab1 regardless of the source of lepidopteran midgut enzymes. Vip3Ab1 was processed at 209Asp as expected based on the location of the 205KVKK↓D cleavage motif. Analysis of earlier time points (<6 hr) indicated the larger molecular weight protein at ~85kDa was processed at 21Ala. This was also the N-terminus of the lower molecular weight product at ~20kDa. Taken together, this suggests stepwise proteolysis where a small amount of the N-terminus is removed prior to further processing. Vip3Bc1, which lacks a conserved dibasic cleavage site, was processed at 214Glu. Similarly to Vip3Ab1, N-terminal sequencing of the ~85kDa protein at early time points indicated that a small portion of the N-terminus was removed at 21Ala. Next, chimeric Vip3 proteins were analyzed after incubation overnight with H. zea midgut extracts (Fig. 4, bottom panel). SDS-PAGE analysis showed the production of similar molecular weight products with Vip3_AB, which was processed at 21Ala and 209Asp as anticipated (Fig. 4, bottom panel). However, Vip3_BA was almost completely degraded to low molecular weight products of <10kDa.
We next investigated the existence of Vip3 oligomers by analytical size exclusion chromatography (SEC; Fig. 5 panels A and B). Vip3Ab1 and Vip3Bc1 both migrated with a retention time consistent with a tetramer (10.1min, ~340kDa) and a minor peak found in the void volume, indicating that the majority of Vip3Ab1 and Vip3Bc1 preparations were tetrameric while in solution. The Vip3_AB chimera also appeared tetrameric in native conditions (Fig. 5, panel C). However, the counterpart Vip3_BA chimera was approximately 50% monomeric and 50% tetrameric when analyzed by SEC (Fig. 5, panel D). With exception of the Vip3_BA chimera, the retention time consistent with a tetramerization persisted even after overnight processing with H. zea midgut enzymes. Collected fractions confirmed processing of Vip3 proteins (not shown). Interestingly, time course SEC analysis of Vip3_BA indicated that monomeric forms of this protein were completely processed to lower molecular weight products (<30kDa) within 45minutes as estimated by retention time (Fig. 6). However, the tetrameric portion of the Vip3_BA chimera was more resistant to degradation by midgut proteases which required 17hours for complete degradation (Fig. 6).
Lastly, we characterized the stability and toxicity of the C-terminal region of Vip3Ab1 and Vip3Bc1. Each protein, beginning at Leu222 (numbering based on Vip3Bc1 sequence) was expressed and purified from recombinant P. fluorescens strains. When analyzed by SEC, both proteins eluted as dimers with an estimated molecular weight of ~120kDa. However, the C-terminus of Vip3Bc1 resolved as a single band at ~60kDa by SDS-PAGE analysis. Interestingly, the Vip3Ab1 C-terminal protein migrated as two different species with molecular weights of 120kDa and 60kDa (Fig. 7). Both proteins were rapidly degraded by CEW gut enzymes and neither demonstrated growth inhibition or lethality against susceptible lepidopteran pests.
Vip3 insecticidal proteins are attractive for the development of transgenic insect-resistant crops owing to their potent and broad spectrum activity against lepidopteran pests. Extant data indicates that Vip3 proteins do not bind to the same receptors as typical crystalline three-domain Bt proteins expressed in transgenic crops19–21. However, information on structure and function of the Vip3 proteins is lacking. Our work has focused on providing fundamental information on the functional domains of a Vip3Ab1, as well as Vip3Bc1, a novel representative of the less characterized Vip3B subfamily.
Vip3Ab1 and Vip3Bc1 are 75% identical from amino acids 1-222 and 54% identical from amino acids 223–812. As noted by other groups, the majority of Vip3 diversity is found at the C-terminus, which has been suggested to be critical for insecticidal activity and specificity5, 22. Vip3Ab1 has lethal activity on H. zea, S. frugiperda, and P. includens. This is not surprising, as several members of the Vip3A family have potent activity within the Spodoptera or Helicoverpa genus of insects7, 12, 14, 22, 23. Also in agreement with previous Vip3A work, Vip3Ab1 lacks insecticidal activity against O. nubilalis. In this study, we show that Vip3Bc1 has insecticidal activity against O. nubilalis and no activity on H. zea or S. frugiperda. Rang et al.18 observed growth inhibition of O. nubilalis by Vip3Ba1, although, no mortality was observed. Therefore, Vip3Bc1 represents the second member of the Vip3B subfamily with insecticidal activity against O. nubilalis. Thus, Vip3Ab1 and Vip3Bc1 have differential insecticidal spectra and are good candidates to evaluate structure –function relationships compared to Vip3A proteins.
Yu et al.14 have shown that Vip3Aa is readily cleaved by midgut proteases from both susceptible and non-susceptible insects and so concluded that toxicity was determined by insect-specific receptor binding. Lee et al. followed on this hypothesis and employed an intact midgut voltage clamp assay to show that only processed Vip3A resulted in pore formation in midguts from susceptible, but not non-susceptible, insects12. Taken together, the studies indicate a minimal requirement for processing, but still support the requirement to bind to a specific receptor. Therefore, we investigated Vip3 processing using midgut enzymes from H. zea and P. includens. Enzymatic digests were carried out at pH 10 to mimic the alkaline pH of the lepidopteran gut. In these conditions, Vip3Bc1 was processed at a much slower rate than Vip3Ab1 by H. zea and P. includens gut enzymes. This is an intriguing finding as P. includens is susceptible to Vip3Bc1 despite relatively slow midgut protease processing. The slow rate of digestion may be due to the lack of a canonical serine protease cleavage site. Based on these data, we cannot conclude if processing is an absolute requirement for toxicity by Vip3B family members. However, it is possible that membrane-bound proteinases not present in our soluble gut enzyme preparations are responsible for processing of Vip3B toxins in vivo.
In this study, we observed an early (~30min) initial cleavage event that occurred with both Vip3Ab1 and Vip3Bc1 at 13ALPSF prior to subsequent downstream cleavage events. This first step in processing removes the signal peptide from the N-terminus. It has been noted that Vip3 proteins are secreted by native Bt cells with putative N-terminal secretion peptides intact3. Furthermore, Selvapandiyan et al.24 reported that removal of the N-terminal 39 amino acids from Vip3Aa9 has no effect on toxicity towards Chilo partellus but had markedly reduced activity towards Spodoptera litura. Other research indicates that N-terminal extension of Vip3 proteins affects the rate of processing and insect activity25. As the 21ALPSF site is well-conserved amongst all current subfamilies of Vip3 proteins, this primary processing step may be an important step common to Vip3A and Vip3B subfamilies that could be involved in maintaining a stable active conformation.
The second proteolytic step occurs in a region between amino acids ~200–220 of both Vip3 proteins in this study (see Fig. 8). Vip3Ab1 is cleaved after 205KVKK↓DSSP as expected, while Vip3Bc1 is processed at 210NVTK↓EVIE when digested with lepidopteran midgut enzymes. Motifs rich in basic amino acids such as arginine or lysine are often sites of serine protease recognition as in the case of proprotein convertases26. Therefore, processing after KVKK is anticipated considering the rich serine protease activity of the lepidopteran gut environment27. However, the Vip3Bc1 processing site was not predicted owing to the lack of an obvious cleavage motif. Vip3Bc1 rate of cleavage at 210NVTK↓EVIE is relatively slow compared to Vip3Ab1, which may be indicative of a less preferred yet exposed protease-sensitive site within the Vip3 structure. Additionally, this region may contain cleavage motifs favored by other proteinases.
Structural information for Cry1 and Cry2 Bt insecticidal proteins has helped define the mechanism of action and provided supporting explanation of differences in binding specificity. This information has been important towards evaluating the potential for cross resistance within the Cry1 family of insecticidal proteins. However, similar information has not yet been determined for Vip3 proteins. Kunthic et al. have overcome technical challenges to generate protein suitable for crystallization and have demonstrated Vip3A oligomerization before and after enzymatic processing28. More recent work has utilized transmission electron microscopy to reveal the surface topology of tetrameric Vip3Ag4 before and after processing29. Considering the oligomeric state and interaction between Vip3 domains, we sought to understand the importance of this association and function of these domains before and after processing by midgut enzymes in native conditions. We performed analytical SEC analysis to demonstrate that a member of the Vip3B family, Vip3Bc1, is also tetrameric in native conditions. Additionally, like Vip3Ab1, Vip3Bc1 persists as a tetramer after gut enzyme processing, which indicates that ~65kDa and ~20kDa products remain associated. Interaction between ~65kDa and ~20kDa has been demonstrated with Vip3Aa30, 31. However, this is the first demonstration the Vip3B proteins behave in a similar manner and suggests a common insecticidal mechanism within the Vip3 family. The precise mechanistic role of tetramer formation remains unclear. However, several observations provide insight towards domain function within Vip3 oligomers. First, we have observed the N-terminal portion of both Vip3Ab1 and Vip3Bc1 to be the most proteolytically stable region of the protein. Digestion experiments conducted with CEW and SBL gut enzymes in the presence of SDS show rapid (t1/2<1minute) degradation of the ~65 kDa C-terminal region, while the N-terminal 20kDa region is relatively stable (see Supplementary Fig. S1). Bel et al. (2017) observed that trypsinized full length Vip3Aa was readily degraded in denaturing conditions, but in native conditions the C-terminal domain remained stable30. However, we have expressed and purified the C-terminal domains (in absence of the N-terminal domain) of Vip3Ab1 and Vip3Bc1 and found them to be labile dimers prone to proteolysis as they are rapidly digested in native conditions. Thus, implicating the requirement for the N-terminal domain to maintain stability in proteolytic native environments. Lastly, in preparations of Vip3_BA chimeras containing tetrameric and monomeric species of Vip3_BA, Vip3 chimera monomers were rapidly degraded while tetrameric forms were stable for several hours. This indicates that tetramer formation may be able to stabilize Vip3 proteins in a proteolytic mileu. Also, despite a high degree of conservation within the N-terminal 20kDa portion of Vip3 proteins, our data demonstrates that this domain is not interchangeable and suggests a specific association with the C-terminal region that provides stability and protects against complete proteolysis. Thus, the specific interaction between and amino and carboxy terminal domains appears to be important for oligomerization of Vip3 proteins, which further increases proteolytic stability in an alkaline proteolytic environment such as the lepidopteran midgut.
Functional evaluation of Vip3 proteins indicated a differential spectrum for Vip3Ab1 and Vip3Bc1. Vip3Bc1 was active on O. nubilalis and P. includens. Vip3Ab1 has a broader spectrum and is active on H. zea, P. includens, S. frugiperda, but not O. nubilalis. The Vip3_BA protein chimera, which contains the Vip3Ab1 C-terminal region, impaired the growth of the same pests as Vip3Ab1, indicating that the C-terminal portion of the protein drives specificity. This region of the Vip3A protein family spanning amino acids ~500–650 contains a predicted carbohydrate binding motif and is therefore thought to play a functional role in toxicity5. Additionally, a model of the C-terminal 200 amino acids of Vip3Aa indicates similarity to domain II of 3 domain Cry toxins, which suggests a role for the Vip3 C-terminal domain as a specificity determinant analogous to Cry toxins of known protein structure32–34. However, the lack of lethality indicates that the cognate N-terminal region is also important for full toxic effect. We hypothesize that this is due to increased susceptibility to proteolytic degradation in the midgut as we have observed in vitro. We observed a complete loss of lethality by both Vip3_AB and Vip3_BA chimeras, including on P. includens, an insect that was especially sensitive to Vip3Ab1 and Vip3Bc1. The loss of Vip3_AB activity was an unanticipated finding as this protein formed expected tetramers and was proteolytically stable, two attributes hypothesized to be required for lethal activity. Fang and colleagues22 generated more conservative Vip3A chimeric proteins within the C-terminal 179 amino acid region and noticed an increase in activity towards O. nubilalis, an insect previously non-susceptible to either parent Vip3A protein. However, this group reported an increase in growth inhibition and not lethality. Therefore, we conclude that the tetramers maintained via specific interactions of amino and carboxy terminal domains of Vip3 proteins not only provides proteolytic stability, but are also required to maintain the specificity determinants required for toxicity.
This study reveals a unique and necessary interaction between the N-terminal and C-terminal regions of two different members of the Vip3 insecticidal protein family. Therefore, our data do not support the existence of a “toxic core”. The majority of the extant literature has not directly inspected the activity of the C-terminal ~65kDa fragment, as experimentation has been performed with full length Vip3A proteins that have been processed in vitro by trypsin7, 8, 11, 19, 31, 35 or isolated lepidopteran gut enzymes12, 36, 37. The insecticidal “core” has been inferred from the conserved nature of the N-terminal ~200 amino acids, the obvious presence of a large ~65kDa fragment after processing, and the large collection of data on 3-domain Bt crystal toxins which demonstrate complete degradation of the C-terminal pro-domain in these proteins38. To our knowledge, two groups have expressed and purified the C-terminal portion of Vip3 from bacterial systems39, 40. Gayen et al. (2012) reported improvements in potency when the N-terminal 200 amino acids were deleted from the expressed Vip3A construct and later demonstrated that this truncated protein could confer protection against Helicoverpa armigera, Agrotis ipsilon, and Spodoptera littoralis in model plant systems. In contrast, others have observed complete loss of activity when the N-terminal 200 amino acids were omitted from another Vip3A expression construct40. They concluded that lack of activity was likely due to a chaperone-like function of the N-terminal ~200 amino acids required for proper folding and proteolytic stability of the C-terminal region. Our data aligns well with those of Li et al.40 as we report similar data with the ~65 kDa C-terminal region of Vip3Ab1 and Vip3Bc1. However, our work with Vip3 chimeras indicates that C-terminal portion of Vip3Bc1 is folded properly, forms tetramers, and is resistant to proteolysis when expressed as a chimera with the Vip3Ab1 N-terminus. However, the Vip3_AB chimera shows complete loss of activity against the four lepidopteran insects tested in this study; H. zea, O. nubilalis, S. frugiperda and P. includens. Thus, we propose that Vip3 proteins do not contain a toxic core similar to other Bt toxins, but rather require a proteolytic activation step that facilitates a change in conformation between ~20kDa and ~65kDa products that is important for stability and specificity.
In conclusion, the Vip family of Bt insecticidal proteins has become an attractive additional option to Bt crystal proteins for expression in crops to endow protection against a broad spectrum of lepidopteran pests. This is evidenced by the commercial success of transgenic Viptera™ corn and VipCot™cotton, which have both been engineered to produce Vip3Aa proteins. Despite the demonstrated agronomic value, little work has been done to understand the commercial potential and biochemical nature of the Vip3 protein family. In this work, we characterized 2 novel insecticidal proteins; Vip3Ab1 and Vip3Bc1. Through this work, we have 1) uncovered a critical role in maintaining protein stability for a the highly conserved N-terminal region, 2) validated the C-terminal region as the major determinant of specificity, and 3) demonstrated a specific interaction between N-terminal and C-terminal regions that permits the formation of a lethal toxin. We believe these studies will serve as a foundation towards a more thorough understanding of the Vip3 family of proteins.
Vip3 genes used in this work,vip3Ab1 (GenBank accession AAR40284) and vip3Bc1 (GenBank accession MF543028), were codon biased for expression in maize. These 2 genes were synthesized and used as templates for amplification. PCR amplification was completed using the Platinum Taq DNA Polymerase (Thermo Fisher, Waltham, MA) and the manufacturer’s protocol. Primers were designed to amplify parts from Vip3Ab1 and Vip3Bc1 to create chimeras by homologous recombination (see Supplementary Table S2). PCR amplicons were cleaned using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and the manufacturer’s protocol. Construction strategy for the parts is shown in Supplementary Table S3. Parts were combined into an E. coli backbone for verification. The chimeras were each sequence verified using Sanger sequencing (Eurofins Genomics, Louisville, KY). Once verified the chimeras were digested from the E. coli backbone using restriction enzymes SpeI and SalI (New England Biolabs, Ipswich, MA) to be ligated into a compatibly digested Pseudomonas flourescens (Pf) expression vector. Ligation was performed using T4 DNA Ligase (New England Biolabs, Ipswich, MA) following the manufacturer’s protocol with 50ng of the digested GOI and 25ng of the Pf expression vector. The ligation mixture was transformed into Pf competent cells and the final constructs were validated via multiple restriction digestions.
All Vip proteins were expressed in recombinant Pseudomonas fluorescens strains as described previously41. Expression of vip genes from the Ptac promoter was induced by addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) after an initial incubation of 24hours at 30°C with shaking in M9 medium supplemented with 1% glucose, trace elements and 5% glycerol. Harvested P. fluorescens cells were sonicated in lysis buffer consisting of 50mM sodium phosphate (pH 8.0), 5% glycerol, 5mM benzamidine HCl, 5mM TCEP and 2mM EDTA. The extract was centrifuged at 20,000 × g for 60minutes. The soluble protein in the supernatant was precipitated with 50% ammonium sulfate and centrifuged at 20,000 × g for 20minutes. The pellet was resuspended in 50mM sodium phosphate (pH 8.0) and purified by anion exchange chromatography using a HiTrap™ Q HP 5mL column with an AKTA Purifier chromatography system (GE Healthcare, UK). The column was equilibrated in 50mM sodium phosphate(pH 8.0), and proteins were eluted with a stepwise gradient to 1M NaCl. Protein-containing fractions were combined and concentrated using Amicon Ultra-15 Centrifugal Filter Devices with a 30kDa MWCO (EMD Millipore). Proteins were desalted to 50mM sodium phosphate (pH 8.0) for bioassay using Zeba® Spin Desalting Columns, 7 MWCO (Thermo Scientific, Waltham, MA). Total protein concentrations were measured with the NanoDrop 2000C Spectrophotometer (Thermo Scientific, Waltham, MA), using the A280 method.
SDS-PAGE analysis was performed using NuPAGE® Novex 4–12% Bis-Tris Protein Gels (Thermo Scientific, Waltham, MA). Proteins were diluted 4X in NuPAGE® LDS Sample Buffer (Thermo Scientific, Waltham, MA) containing 100mM TCEP prior to loading onto the gel. Ten µL of Novex Sharp Pre-stained Protein Standard was loaded onto one lane of each gel. Gels were run according to the manufacturer’s recommendations using NuPAGE® MES SDS Running Buffer and stained with SimplyBlue SafeStain, then destained in water and imaged on a flatbed scanner.
Samples were prepared for N-terminal sequencing by blotting an SDS-PAGE gel onto a PVDF pre-cut blotting membrane (Thermo Scientific, Waltham, MA) via wet tank transfer in 10mM CAPS (pH 11) with 10% methanol. The PVDF membrane was stained for 15–20seconds with Coomassie Brilliant Blue (Bio-Rad, Hercules, CA), destained in 45% methanol; 10% acetic acid, rinsed with water and air dried. The target bands were excised and analyzed by Edman degradation using a Shimadzu PPSQ-33A protein sequencer (Shimadzu, Kyoto, Japan). The data was analyzed with Shimadzu data analysis software.
Midguts were dissected from insects and placed into vials containing 8.5% sucrose and 150mM NaCl. Soluble proteins, including digestive proteases, were isolated by centrifugation for 30minutes at 10,000 x g. Proteolytic activity was normalized using BODIPY-casein degradation assay. In this assay, fluorescein labeled casein is incubated at 10µg/mL 50mM 3-(N-morpholino)propanesulfonic acid (MOPS) with extract and fluorescence is monitored over time as relative fluorescence units per second (RFU). Vip proteins were digested with Corn Earworm (Helicoverpa zea) or Soybean Looper (Pseudoplusia includens) gut fluid at 4µL/mL and 8µL/mL, respectively. Proteins were added to the reaction for a final concentration of 150µg/mL in 50mM MOPS pH 10 buffer. Control reactions were prepared containing no insect gut fluid, except for size exclusion controls, which contained gut fluids inactivated by heating at 95°C for 15minutes. Reactions were incubated at 30°C with shaking for varying time intervals. Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO) was added to terminate the reactions prior to SDS-PAGE and size exclusion analysis.
All analyses were performed on an Agilent 1200 HPLC (Agilent, Santa Clara, CA) using a TSKgel SuperSW3000 2.0mm×30cm, 4µm column (Tosoh Biosciences Tokyo, Japan) isocratically in 50mM sodium phosphate pH 7.0 containing 250mM NaCl at a flow rate 75µl/min for 30min. Injection volumes were 3µl at a protein concentration of 150µg/ml. Protein molecular weight standards were purchased from Sigma (#69385) and reconstituted according to the manufacturer’s directions. Fluorescence detection was monitored at 295nm excitation and 345nm emission. Data analysis was performed using ChemStation software (Agilent).
Proteins were tested for insecticidal activity using neonate Lepidopteran larvae on artificial insect diet. Larvae of H. zea, O. nubilalis, S. frugiperda and P. includens were hatched from eggs obtained from Benzon Research Inc. (Carlisle, PA). The bioassays were conducted in 128-well plastic trays specifically designed for insect bioassays (C-D International, Pitman, NJ). Each well contained 1.5mL of Multi-species Lepidoptera diet (Southland Products, Lake Village, AR). A 40µL aliquot of protein sample was delivered by pipette onto the 2cm2 diet surface of each well (20μL/cm2). Treatment concentrations were calculated as the amount (ng) of protein per square centimeter (cm2) of surface area in the well. The treated trays were held in a fume hood until the liquid on the diet surface had evaporated or was absorbed into the diet. Within a few hours of eclosion, individual larvae were picked up with a moistened camel hair brush and deposited on the treated diet, one larva per well. Sixteen animals were used per treatment. The infested wells were then sealed with adhesive sheets of clear plastic, vented to allow gas exchange (C-D International, Pitman, NJ). Bioassay trays were held at 28°C, ~40% relative humidity and 16:8hours light:dark for 5 days, after which the total number of insects exposed to each protein sample, the number of dead insects and number of moribund insects were recorded. Moribund insects were classified as insects that were still alive, did not increase in size over the course of the bioassay, and did not respond to perturbation.
Statistical analysis was carried out using JMP® Pro Version 11 software (SAS Institute Inc., Cary, NC). Lethal concentrations (LC50) of Vip3 proteins were calculated on sum of dead and moribund insects using a generalized linear model utilizing Probit analysis of binomial data. An inverse prediction of LC50 with 95% confidence intervals was calculated based on this model. For determination of growth inhibition, total live insect mass was weighed after 5 days of treatment and normalized for insect number. Sixteen insects were used per treatment and experiments were repeated twice. Insects tested on buffer control diet were compared to Vip3-treated samples. Average weight of insects after Vip3 treatment was modeled using linear mixed models for each insect species respectively:
where Y is the insect average weight observed in each treated well, µ is the overall mean, T is the effect of Vip3 protein at a specific dose, E is the effect of different experimental run, is the error. The Vip3 protein treatment effect T was modeled as a fixed effect, while experimental run E was treated as a random effect. Box-Cox transformation was applied to correct for non-normality and heterogeneous variances. Average insect weight was compared with null hypothesis of no growth difference between a Vip3 protein treatment and the untreated control (buffer) and the alternative hypothesis of different growth under a Vip3 protein treatment. We used Dunnett’s test to adjust P-values for multiple comparisons.
All data generated and analysed during this study are included in this published article.
This work was supported by Dow AgroSciences. The authors would like to thank Terry Walsh and Steve Evans for critical review of this manscript.
M.Z. and K.N. conceived the study. M.Z., M.S., M.F., S.Y.T., J.A., and T.L. designed and performed the experiments. M.Z., S.Y.T., and X.W. analyzed the data. M.Z. wrote the manuscript. All authors reviewed the mansucript.
The authors declare that they have no competing interests.
Electronic supplementary material
Supplementary information accompanies this paper at doi:10.1038/s41598-017-11702-2
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