Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests

Journal of Invertebrate Pathology 149 (2017) 59–65

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Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests

MARK

Wen-bo Chena,b, Guo-qing Luc, Hong-mei Chengc, Chen-xi Liub, Yu-tao Xiaob, Chao Xud, ⁎ Zhi-cheng Shend, Kong-ming Wub, a Fujian Provincial Key Laboratory of Insect Ecology, Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China b The State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China c Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China d Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, Zhejiang, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Bacillus thuringiensis Vegetative insecticidal proteins Midgut putative receptor Transgenic cotton Spodoptera spp

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for integrated pest management in China.

1. Introduction Bacillus thuringiensis is a gram-positive bacterium that produces insecticidal crystal proteins (Cry) during its sporulation phase (Schnepf et al., 1998). The Cry proteins or the delta-endotoxins are the most well-known insecticidal proteins and have been extensively used in sprays and transgenic crops to control major agricultural lepidopteran pests such as cotton bollworm (Helicoverpa armigera), tobacco budworm (Heliothis virescens), European corn borer (Ostrinia nubilalis) and pink bollworm (Pectinophora gassypiella) (Carrière et al., 2003; Wu and Guo, 2005, Wu et al., 2008; Adamczyk and Hubbard, 2006). They are harmless to vertebrates and most other organisms (Mendelsohn et al., 2003; Sanahuja et al., 2011; Comas et al., 2014; Nicolia et al., 2014). Since 1997, farmers in China have grown transgenic Bt-Cry1Ac cotton to control the major pest H. armigera and have achieved great success.

⁎

However, Bt-Cry1A toxins do not target all lepidopteran insect pests; many secondary lepidopteran pests such as the common cutworm (Spodoptera litura), beet armyworm (Spodoptera exigua), and black cutworm (Agrotis ipsilon), which are relatively nonsusceptible to Cry1Ac, have caused considerable economic damage, and pesticides and/or other control strategies have been needed to control these pests (Adamczyk et al., 1998, 2008; Cui et al., 2002; Wan et al., 2008; Wolt, 2010; Akin et al., 2011). There is even a risk that S. litura could become an alarming, major pest of Bt-Cry1Ac cotton in the Yangtze River Valley of China (Wan et al., 2008). Thus, new insecticidal proteins are necessary to manage and control secondary pests that are becoming rampant in China. In addition to Cry proteins, B. thuringiensis synthesizes vegetative insecticidal proteins (Vips) during its vegetative growth phase. Since Vip3 was first isolated from B. thuringiensis in 1996 (Estruch et al.,

Corresponding author. E-mail addresses: [email protected] (W.-b. Chen), [email protected] (G.-q. Lu), [email protected] (H.-m. Cheng), [email protected] (C.-x. Liu), [email protected] (Y.-t. Xiao), [email protected] (C. Xu), [email protected] (Z.-c. Shen), [email protected] (K.-m. Wu). http://dx.doi.org/10.1016/j.jip.2017.08.001 Received 10 April 2017; Received in revised form 25 June 2017; Accepted 1 August 2017 Available online 04 August 2017 0022-2011/ © 2017 Elsevier Inc. All rights reserved.

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gene generated by an overlap method, this chimeric protein consists of the N-terminal 600 amino acid residues of Vip3Ac1 (GenBank accession DQ054848) and the C-terminal 189 amino acid residues of Vip3Aa1 (GenBank accession L48811) (Fang et al., 2007). It was modified to encode a six his-tag sequence at the N- and at the C-terminus. After it was subcloned into expression vector pET28a, it was inserted into Escherichia coli strain BL21 Star (Stratagene). Vip3AcAa protein expression was induced with 1 mM isopropy-β-d-1-thiogalactopyranoside (IPTG) for approximately 15 h at 20 °C in a shaking incubator. For purification of the expressed Vip3AcAa, cultured E. coli BL21 cells were collected by centrifugation at 3000g for 15 min at 4 °C, washed with 50 ml of ice-cold PBS buffer, resuspended in 30 ml of PBS buffer, and sonicated for 15 min on ice. After centrifugation at 25,000g for 20 min at 4 °C, the soluble fraction including expressed Vip3AcAa was subjected to affinity purification using Ni-Sepharose beads (Amersham Biosciences, USA) and dialyzed against PBS buffer. The concentration of Vip3AcAa protoxin and the activated toxins was assessed by densitometry with a VersaDoc imaging system and Quantity One image analysis software (Bio-Rad Laboratories, USA) after SDS–PAGE, using a set of known bovine serum albumin (BSA) solutions as standards. The proteins were stored at −80 °C until used.

1996), more than 100 types of Vip and Vip-related sequences have been recorded and classified into four groups (Vip1 to Vip4), based on the amino acid sequence similarity (Crickmore, 2017). Vip1 and Vip2 must combine to form a binary toxin to be toxic, and their combination has been reported to display insecticidal activity against 10 coleopteran species and the homopteran species Aphis gossypii (Sattar and Maiti, 2011; Shingote et al., 2013; Bi et al., 2015; Welch et al., 2015). No target insects have yet been identified for Vip4 proteins. Vip3 proteins are currently classified into three classes (Vip3A, 3B, and 3C) based on their amino acid identity, and compared with Cry proteins, Vip3A proteins have a broad insecticidal spectrum against a large number of lepidopteran species; pests that are relatively tolerant to Cry proteins are highly susceptible to Vip3A proteins (Estruch et al., 1996; Yu et al., 1997; Zhu et al., 2006; Chakroun et al., 2012, 2016; HernándezMartínez et al., 2013; Chakroun et al., 2016). The mode of action of Cry1A toxins have been well studied (Jurat-Fuentes and Adang, 2006; Bravo et al., 2007; Flores-Escobar et al., 2013; Pardo-López et al., 2013), but knowledge of the underlying mechanism of Vip3A remains to be clarified. Although its mechanism has some commonality with the mode of action of Cry1A toxins, its specific receptors differ from the known Cry toxin binding receptors, and it forms ion channels that are distinct from those of Cry1A, and thus presumably differs in its mode of action (Lee et al., 2003, 2006; Abdelkefi-Mesrati et al., 2011b; Bergamasco et al., 2013; Hamadou-Charfi et al., 2013). Therefore, the Vip3A proteins could be a viable candidate for developing a broadspectrum toxin against lepidopteran pests. In fact, Syngenta Biotechnology successfully introduced the gene vip3Aa19 into cotton (COT102) and vip3Aa20 into corn (MIR162). Then both were pyramided with cry1Ab gene (VipCot Vip3Aa plus mCry1Ab in cotton; Agrisure Viptera Vip3Aa + Cry1Ab in corn) and later with cry1Fa (VipCot Vip3Aa plus Cry1Ac plus Cry1Fa in cotton; Agrisure Viptera Vip3Aa plus Cry1Ab plus Cry1Fa in corn) to confer wider protection against various lepidopterans and delay the development of resistance in insects (Carrière et al., 2015; Chakroun et al., 2016). Furthermore, corn was pyramided with Cry1Ab + Cry1Fa + Vip3Aa + mCry3A + eCry3.1Ab to confer protection against coleopteran pests (Carrière et al., 2015; Chakroun et al., 2016). The newest Bt cotton “pyramid”, Cry1Ac + Cry2Ab + Vip3Aa, is scheduled for commercial release in Australia and the United States (Carrière et al., 2016). In a previous study, we used an overlap method to develop a Vip3Ac and Vip3Aa chimeric protein, Vip3AcAa (GenBank accession KX345937) (Fang et al., 2007). This chimeric protein is insecticidal to O. nubilalis (O. nubilalis is tolerant to almost all known Vip3A proteins), exerted high toxicity to the fall armyworm (Spodoptera frugiperda) and is highly insecticidal to a Cry1Ac-resistant strain of cabbage looper (Trichoplusia ni) (Fang et al., 2007). In the present study, we tested the insecticidal activity of this chimeric protein against S. litura, S. exigua, and A. ipsilon. To further understand the mechanism of Vip3AcAa against these agronomically important pests, we investigated potential specific-binding sites in brush border membrane vesicle (BBMV) of these lepidopteran pests. Finally, insect resistance of the pyramided Vip3AcAa + Cry1Ac cotton was tested against S. litura, S. exigua, and A. ipsilon. Our results indicated that Vip3AcAa protein had high insecticidal activity against these three pests and that the biotinylated Vip3AcAa protein specifically bound to their BBMV; BBMV protein blotting experiments showed the potential receptors. Moreover, the use of this new transgenic cotton line expressing Vip3AcAa and Cry1Ac will enhance the potency of insect resistance cotton and expand the insecticidal spectrum against secondary pests that are relatively tolerant to Cry1A proteins.

2.2. Insect rearing and bioassays We used a surface contamination bioassay to determine the toxicity of Vip3AcAa protoxins, trypsin-activated Vip3AcAa and the Cry1Ac protoxins against S. litura, S. exigua and A. ipsilon, which had been collected from fields and reared for several years in our laboratory on an artificial diet without exposure to any Bt toxins. All the insects were cultured in our insectary at 27 ± 2 °C and 75 ± 10% relative humidity (RH) with 14 h light/10 h dark. The Cry1Ac protoxins were kindly supplied by Biotechnology Research Laboratory, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS). Testing at least seven concentrations of toxins in all bioassays in our insectary, we put one first instar larva in each well of a 24-well plate, with 24 first instars in each replicate and three replicates per treatment (total n = 72 per treatment). Larvae that died or did not reach the third instar within 7 days were considered “effectively dead”. The LC50 in dose-response bioassays was estimated using POLO-PC software (Russell et al., 1977). 2.3. Preparation of BBMV, Vip3AcAa labeling and binding assays BBMV was prepared from last instar larvae of those three lepidopteran pests using differential magnesium precipitation (Wolfersberger et al., 1987), then kept at −80 °C until used. The protein concentration was determined by the Bradford (1976) method with BSA as the standard. Purified Vip3AcAa protoxins were digested by 5% (wt/wt) commercial trypsin at 37 °C for 3 h. The reaction was terminated by addition of 1 × EDTA-free Complete Protease Inhibitor Cocktail (Roche, Germany). The trypsinized Vip3AcAa toxin was labeled with biotin using a Biotin Labeling Kit (Elabscience, China) and the manufacturer’s instructions. The labeled Vip3AcAa was quantified as described above. For competition experiments, labeled Vip3AcAa toxin (0.1 µg) was incubated with BBMV (25 µg) in PBS buffer, and the reaction mixture was incubated on a rotator for 1 h at room temperature in the absence or presence of unlabelled Vip3AcAa toxins (5-, 25-, and 100-fold) or 100-fold excess of unlabeled activated Cry1Ac. Subsequently, unbound toxin in the supernatant was removed by centrifugation at 15, 000g for 30 min at 4 °C, and pellets were washed two times with PBS buffer. The pellets were then suspended in 15 µl of PBS and separated using 4–20% gradient SDS–PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was blocked for 2 h at room temperature in blocking buffer (PBST buffer [PBS buffer, 0.1% Tween-20], 3% BSA, pH 7.4). Labeled Vip3AcAa that had bound to

2. Materials and methods 2.1. Expression and purification of Vip3AcAa The vip3AcAa gene (GenBank accession KX345937) is a chimeric 60

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molar concentrations (Supplementary Table S1), and there was not any significant difference between the insecticidal activities of the protoxin and the activation pretreatment of Vip3AcAa against S. litura, S. exigua and A. ipsilon, on the basis of the conservative criterion of non-overlap between the 95% fiducial limits (Supplementary Table S1), indicated that the protease treatment has not a significant influence on the susceptibility.

BBMV was visualized by incubating the membrane with horseradish peroxidase (HRP)-conjugated streptavidin (Thermo Fisher Scientific, USA) (1:2000 dilution) for 2 h at room temperature, the washed five times for 5 min each in PBST buffer. Binding was visualized using Super ECL Plus Detection Kit (Applygen, China) and the Image Quant LAS4000 image analyzer (GE Healthcare, Sweden). 2.4. Ligand blot

3.2. Binding assays Ligand blot analysis was used to identify the putative trypsinized Vip3AcAa toxin binding receptors on BBMV. BBMV proteins (15 µg) were separated using 4–20% gradient SDS–PAGE and transferred onto a PVDF membrane. The PVDF membrane was blocked for 2 h with 20 ml blocking buffer (PBST buffer, 3% BSA, pH 7.4), then incubated in PBST buffer containing 15 nM of labeled Vip3AcAa toxin for 1 h. After five 5min washes in PBST buffer, the PVDF membrane was incubated with HRP-conjugated streptavidin (1:8000) for 1 h at room temperature in PBST buffer, and visualized as described above. PageRuler (Thermo Fisher Scientific, USA) pre-stained standard protein marker was used, and Quantity One image analysis software was used to estimate the molecular weights of the bands.

To determine whether trypsinized Vip3AcAa specifically binds to BBMV, we used homologous unlabeled Vip3AcAa and heterologous, unlabeled, activated Cry1Ac to compete with the labeled Vip3AcAa. As shown in Fig. 1, with an increase in unlabeled Vip3AcAa (5-, 25-, and 100-fold), the amount of labeled Vip3AcAa binding to BBMV decreased significantly, indicating that the interaction between labeled Vip3AcAa and BBMV was inhibited by the unlabeled Vip3AcAa. However, the inhibition was not significant with a 100-fold excess of activated Cry1Ac. These results indicate that Vip3AcAa can specifically bind to BBMV, and Vip3AcAa does not share the same binding sites with activated Cry1Ac in the BBMV.

2.5. Cotton lines and insect-resistance bioassay

3.3. Ligand blot

Cultivar line CV163 that expresses Vip3AcAa and Cry1Ac was developed by the Biotechnology Research Institute (CAAS) using the Agrobacterium-mediated method. T2, T3 generation of CV163 and its nontransformed parental line Coker 312, and NuCOTN33B (33B) expressing only Cry1Ac, were planted at the Langfang Experimental Station of the CAAS, Hebei Province, China in 2015 and 2016. At the 2–3 true-leaf stage, the CV163 plants were sprayed with 2 g/kg kanamycin to cull negative segregating plants. The remaining plants were grown using standard practices without insecticides. PCR, western blot (for Vip3AcAa protein) and ELISA (for Cry1Ac protein) were used to further determine positive plants (Supplementary Fig. S1). Newly unfurled leaves (3rd node from the top of the plant) collected from field-grown cotton 70 days and 85 days after planting in 2015 and 2016, respectively, were used to evaluate the toxicity of non-Bt, Bt cotton that only expressed Cry1A, and CV163 that expressed Vip3AcAa and Cry1Ac against S. litura, S. exigua and A. ipsilon. Neonatal larvae were placed on leaf lettuce in a 500 ml glass cup and allowed to feed for 24 h before being transferred onto cotton leaves (five larvae per leaf). The leaf stalk of the detached cotton leaves was inserted into 2% agar in a glass tube (12 cm in height, 3.3 cm in diameter) with a cotton ball at the top to keep larvae from escaping. The tubes were then put in the insectary at 27 ± 2 °C, 60 ± 10% RH, and 14 h light/10 h dark. Then 20 or 25 glass tubes with a cotton leaf were arbitrarily divided into 4 or 5 groups (n = 5 cups per group, total 100 or 125 neonates per combination of cotton cultivar and pest species). Larvae were considered dead if stimulation with blunt tweezers did not elicit a coordinated response after 5 days. Significant differences in larval mortality among different cotton cultivars were compared using one-way ANOVA and Tukey’s honestly significant difference [HSD], (P < 0.05).

To identify potential Vip3AcAa protein-binding receptors present in the midgut BBMV of S. litura, S. exigua and A. ipsilon, we measured the in vitro binding of Vip3AcAa protein to the BBMV proteins separated by SDS-PAGE. As shown in Fig. 2, labeled Vip3AcAa bound to four putative receptors of approximately 36, 39, 68, and 100 kDa on the BBMV from S. litura (Fig. 2A), two putative receptors of approximately 34 and 100 kDa on the BBMV from S. exigua (Fig. 2B), and three putative receptors of approximately 35, 55, and 110 kDa on the BBMV from A. ipsilon (Fig. 2C). 3.4. S. litura, S. exigua and A. ipsilon were susceptible to transgenic cotton that co-expressed Vip3AcAa and Cry1Ac Larval mortalities for S. litura, S. exigua and A. ipsilon were not significantly higher when feeding on the Bt cotton NuCOTN33B (33B) that only produced Cry1Ac (Bollgard) compared with larvae feeding on non-Bt cotton (Coker312), indicating that 33B cotton was not protected from these three pests (Fig. 3). Larval mortalities were significantly higher when fed on the new transgenic line CV163 that expressed Vip3AcAa and Cry1Ac compared with those that fed on 33B or Coker312 in the laboratory tests (75 ± 8.06% and 64 ± 4.20% larvae of S. litura, 78 ± 5.29% and 59 ± 7.72% larvae of S. exigua, 70 ± 5.31% and 59 ± 4.63% larvae of A. ipsilon were killed when fed on CV163 cotton after 5 days in 2015 and 2016 year, respectively), indicating that the CV163 line has potential for protecting cotton from damage by these secondary pests that are relatively tolerant to Cry1Ac proteins (F2,9=53.41, P = 0.0001; F2,11 = 65.00, P = 0.0001; against S. litura in 2015 and 2016 year, respectively) (F2,9 = 76.46, P = 0.0001; F2,9 = 13.55, P = 0.0019; against S. exigua in 2015 and 2016 year, respectively) (F2,12 = 20.48, P = 0.0001; F2,12 = 29.35, P = 0.0001; against A. ipsilon in 2015 and 2016 year, respectively).

3. Results

4. Discussion

3.1. Vip3AcAa insecticidal activity against larvae of S. litura, S. exigua and A. ipsilon

We investigated the insecticidal activities of Vip3AcAa protoxin, trypsin-activated Vip3AcAa toxin, and Cry1Ac protoxin against first instar larvae of S. litura, S. exigua, and A. ipsilon. The low Cry1Ac toxicity to these pests in our bioassays agrees with previous reports (Adamczyk et al., 1998, 2008; Akin et al., 2011; Hernández-Martínez et al., 2008; Lu et al., 2009). A previous study indicated that S. litura digested Cry1Ab into small fragments (50 kDa) with a non-active form and high excretion of Cry1Ab via feces (Shu et al., 2017). Cry1Ac toxin

Cry1Ac had very low toxicity against S. exigua larvae and negligible toxicity against S. litura and A. ipsilon larvae. The Vip3AcAa had high insecticidal activity against all three lepidopterans. Based on the effective mortality, the LC50 values calculated for the protoxin and activated Vip3AcAa toxin against S. litura were 36 and 23 ng/cm2, 152 and 67 ng/cm2 against S. exigua, and 75 and 28 ng/cm2 against A. ipsilon (Table 1). For a fair insecticidal activity comparison, we converted to 61

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Table 1 Bioassay results for Vip3AcAa and Cry1Ac toxins against larvae of S. litura, S. exigua and A. ipsilon. Proteins

Vip3AcAa protoxin Trypsin-activated Vip3AcAa Cry1Ac protoxin a b c d e

S. litura

S. exigua

A. ipsilon

LC50a (FL95b)

Slope

LC50 (FL95)

Slope

LC50 (FL95)

Slope

36 (27–45) 23 (17–29) > 6000c

1.33 ± 0.13 1.54 ± 0.15 –

152 (111–205) 67 (46–93) > 6000d

1.34 ± 0.09 0.85 ± 0.09 –

75 (51–107) 28 (20–36) > 6000e

0.86 ± 0.10 1.29 ± 0.13 –

Concentration (ng toxin/cm2 diet) killing 50%, based on the effective mortality. 95% fiducial limits (FL95). Maximum concentration tested; mean effective mortality was 8.33% of total larvae. Maximum concentration tested; mean effective mortality was 35.67% of total larvae. Maximum concentration tested; insects were not affected.

Song et al., 2016). In similar bioassays, trypsin-activated Vip3Ae and Vip3Af toxins did not have stronger toxicities compared with the respective protoxins against S. frugiperda and A. ipsilon, respectively (Hernández-Martínez et al., 2013). Similar to the above results, the insecticidal toxicities of Vip3AcAa protoxin and activated toxin for S. litura, S. exigua and A. ipsilon were not significantly different from each other (on the basis of the conservative criterion of non-overlap between the 95% fiducial limits) (Supplementary Table S1). In other independent experiments, a correlation between proteolytic processing and insecticidal toxicities of Vip3Aa protein was identified. The protein activation step involved in differential susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Vip3Aa16 toxin and the subsequent zymogram analysis suggested that the difference between proteolysis products was due to variability in the proteases from the larvae of these species (Abdelkefi-Mesrati et al., 2011a). Moreover, the difference in the activation rates of the Vip3Aa protoxin between S. exigua and S. frugiperda is the basis for differential susceptibility toward this toxin (Chakroun et al., 2012). In the recent report, Bel et al. (2017) found that the largest activated fragment (62–66 kDa) of Vip3A is extremely stable to trypsin or midgut juice proteases. Vip3A protoxin is unfolded at the presence of SDS, along with the low efficacy of trypsin inhibitors to stop the proteolytic reaction, making the SDS-PAGE analysis reveal secondary typsin cleavage sites yielded the band of approximately 29, 32, and 42kDa, which give artefactual band patterns, and even the apparent complete degradation of the Vip3A proteins under denaturing conditions (Bel et al., 2017). So previous published data about the proteolysis of Vip3A proteins might have given the false impression that the Vip3A proteins had been degraded during the proteolytic reactions. Our bioassay results indicated that the protease pretreatment did not have a significant influence on susceptibility against the same pest. Because binding of Cry toxin with receptors plays an important role in the mode of action (Pardo-López et al., 2013), Vip3A binding to the larval midgut epithelium is likely to be important in toxicity. Specificbinding sites of Vip3A protein in midgut membranes of various lepidopteran pests are essential for toxicity, and putative receptors of different molecular weights were identified for the Vip3A toxin in several tested lepidopteran pests (Table 2). So far, a 48-kDa protein from A. ipsilon, called tenascin, has been identified and could be associated with apoptotic processes (Estruch and Yu, 2001). The S2 ribosomal protein from S. litura was also identified as the Vip3A receptor in Sf21 cells (Singh et al., 2010). In our labeled-Vip3AcAa binding assays, the activated Vip3AcAa bound specifically to BBMV of these three pests and did not compete with Cry1Ac. The lack of shared binding sites has also been examined for Vip3A in relation to Cry1Ac, Cry1Ab, Cry1Fa, Cry2Ae, and Cry2Ab in many lepidopteran species (Lee et al., 2006; Sena et al., 2009; Liu et al., 2011; Gouffon et al., 2011; Hamadou-Charfi et al., 2013; Chakroun and Ferré, 2014). In vitro binding of Vip3AcAa protein to BBMV separated by SDS-PAGE, labeled Vip3AcAa putatively bound to approximately 36, 39, 68, and 100 kDa receptors on BBMV in S. litura; approximately 34 and 100 kDa receptors on BBMV in S. exigua; approximately 35, 55, and 110 kDa receptors on BBMV in A. ipsilon;

Fig. 1. Specific binding of labeled Vip3AcAa to brush border membrane vesicle (BBMV). BBMV derived from S. litura (A), S. exigua (B), and A. ipsilon (C); lane 1: labeled Vip3AcAa bound to BBMV (no competitor); lanes 2–4: homologous competition, labeled Vip3AcAa and excess unlabeled Vip3AcAa (5-, 25- and 100-fold, respectively) bound to BBMV; lane 5: heterologous competition, labeled Vip3AcAa and 100-fold excess unlabeled activated Cry1Ac.

Fig. 2. Detection of potential receptors for labeled Vip3AcAa in BBMV. BBMV derived from S. litura (A), S. exigua (B) and A. ipsilon (C); BSA as negative control (D).

is retained by the peritrophic matrix and then excreted by A. ipsilon (Rees et al., 2009). A peritrophic matrix baffle, Cry toxin degradation by midgut proteases and gut defensive responses during ingestion of the toxin could contribute to the lack of susceptibility toward Cry1A toxins in some insects (Rees et al., 2009; Jurat-Fuentes and Crickmore, 2016; Lu et al., 2017; Shu et al., 2017). The Vip3AcAa protoxin and trypsinactivated Vip3AcAa toxin had high insecticidal toxicities against these pests, and the toxicity is not lower than with natural Vip3Aa (Doss et al., 2002; Lee et al., 2003; Gayen et al., 2012; Baranek et al., 2015; 62

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Fig. 3. Larval mortality of S. litura, S. exigua and A. ipsilon on different cultivars of cotton. Insect-resistance bioassay in the laboratory in 2015 (A) and 2016 (B). Different lowercase letters above bars for the same pest species indicate significant differences (one-way ANOVA and Tukey’s honestly significant difference [HSD], P < 0.05).

and expression of Vip3A improved; plants were completely resistance against H. zea, S. frugiperda and S. exigua (Wu et al., 2011). The insecticidal toxicities of Vip3AcAa against S. litura, S. exigua, and A. ipsilon are superior to Cry1Ac toxins, but the insecticidal toxicity of Cry1Ac against H. armigera, P. gossypiella and O. nubilalis are superior to Vip3A toxins. For achieving more durable and broader-spectrum insecticidal activity, we inserted vip3AcAa and cry1Ac into cotton using an Agrobacterium-mediated method and obtained one successful insectresistant cotton line, CV163. Insect-resistance tests indicate that Bt cotton that expressed only Cry1Ac protein does not effectively control S. litura, S. exigua and A. ipsilon, whereas our CV163 line that expressed Vip3AcAa and Cry1Ac has potential for protecting cotton from damage by these secondary, but important cotton pests. We also noted that larval mortalities in 2016 were lower than in 2015 after larvae fed on CV163. We were not able to quantify Vip3AcAa protein expression using the existing Vip3A ELISA kit and could not determine whether differences in mortality were caused by differences in protein expression. But processed during growth and development of transgenic cotton is believed to regulate the expression of the Bt protein, and high temperature, drought and waterlogging, nitrogen nutrition and salinity stress can also affect the efficacy of control and level of Bt protein in transgenic Bt cotton (Luo et al., 2017). Moreover, the CV163 line with pyramided Vip3A and Cry1Ac is a single-trait cotton for these secondary pests, so there is a strong likelihood that secondary pests will become resistant. But stacking other toxins that are toxic to these secondary pests and have a mechanism of action that differs from that of Vip3A could potentially result in a more durable insect-resistant pyramided cotton in the future. Bt cotton has been used as one component in the overall management of insect pests in the diversified cropping systems common throughout China. Our results indicate that Vip3AcAa can be used as a new toxin against lepidopteran pests in transgenic plant breeding for expanding the insecticidal spectrum and complementing current integrated pest management strategies in China. For comprehensively demonstrating that cultivar CV163 can enhance insect resistance in cotton in the field, bioassays and biological safety evaluations in the field are still needed.

Table 2 Molecular weight of putative receptors for Vip3A toxin in different lepidopteran insects. Toxins

Insect species

Molecular weight of putative receptors (kDa)

References

Vip3Aa

M. sexta A. ipsilon P. oleae

80 and 100 48 65

E. kuehniella

65

S. littoralis

55 and 100

S. frugiperda

65

S. albula

65

S. cosmioides

65

S. eridania

65

S. litura S. exigua A. ipsilon

36, 39, 68, and 100 34 and 100 35, 55, and 110

Lee et al. (2003) Estruch and Yu (2001) Abdelkefi-Mesrati et al. (2009) Abdelkefi-Mesrati et al. (2011a) Abdelkefi-Mesrati et al. (2011b) Bergamasco et al. (2013) Bergamasco et al. (2013) Bergamasco et al. (2013) Bergamasco et al. (2013) Present study Present study Present study

Vip3Aa16

Vip3Aa43

Vip3AcAa

suggested that the Vip3AcAa has similar size receptors (34–36 kDa) in these three pests, and they are probably the same type of binding receptors. Future studies to identify potential Vip3AcAa receptors by amino acid sequencing in these pests are critical for clarifying the mode of action of this toxin. Although first-generation Bt cotton that expresses only Cry1Ac or the fusion protein Cry1Ac/Cry1Ab can still effectively protect cotton against the major pest H. armigera in China, the frequency of resistance to Cry1Ac has regularly and significantly increased in populations of H. armigera (Zhang et al., 2011; Tabashnik et al., 2012; Jin et al., 2015), and various Spodoptera species and A. ipsilon are not susceptible to Cry1A toxins. VipCot cotton, that only expresses the Vip3Aa protein, is highly efficacious against H. armigera (Llewellyn et al., 2007), whereas VipCot™ cotton that is pyramided with vip3A and cry1Ab genes confers protection against two other key cotton pests, Helicoverpa zea and Heliothis virescens (Kurtz et al., 2007); furthermore, the modified Vip3A protein introduced into tobacco also confers protection against H. armigera, A. ipsilon and S. littoralis (Gayen et al., 2015). When a synthetic vip3A gene fused to a chloroplast transit peptide coding sequence was inserted into cotton, the Vip3A protein accumulated in chloroplasts,

Acknowledgments This work was funded by grants from the Key Project for Breeding Genetic Modified Organisms (grant no. 2016ZX0812-004) and the 63

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