Indications for the Potential of Cry1Ab1 for Inhibiting Aphid Development and Virus Transmission

doi:10.21203/rs.3.rs-1236673/v1

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Indications for the Potential of Cry1Ab1 for Inhibiting Aphid Development and Virus Transmission

https://doi.org/10.21203/rs.3.rs-1236673/v1

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Cry1A-type toxins are insecticidal proteins from Bacillus thuringiensis that are widely used to control Lepidoptera pests, and they are believed to be useless for the control of Hemiptera pests. In this paper, the purified Cry1Ab1 toxic core rarely killed peach aphids (Myzus persicae), as its Lethal Concentration 50 (LC₅₀) was up to 1308.6 µg/ml, as measured using the membrane-encapsulation method. We then identified the proteins that bind to the Cry1Ab1 toxic core in M. persicae using pull-down assays and liquid chromatography−tandem mass spectrometry (LC-MS/MS), and then analyzed the associated functions of these proteins using the STRING database. GPN-loop GTPase 2, beta-actin, ATP synthase subunit alpha and an unknown and annotated protein, which are mainly involved in cell phagocytosis, RNA polymerase, cellular oxidative phosphorylation and other related functions, were the proteins that bound to the Cry1Ab1 toxic core. Indications from docking showed that Cry1Ab1 toxic core, ATP synthase subunit alpha and beta-actin or Cry1Ab1 toxic core, ATP synthase subunit alpha and GPN-loop GTPase 2 being a complex to exert effects; Finally, models for underlying the molecular mechanism of Cry1Ab1 in M. persicae and disturbing virus transmission were proposed. The study will suggest new insights for aphid-effective Cry evaluation and improvement.

Aphid development

Bacillus thuringiensis

Cry1Ab1

Cry-binding proteins

Mechanism model

Virus transmission

In recent years, the extensive use of Bacillus thuringiensis (Bt) pesticides in China has increased. The total proportion of Bt biopesticides in the microbial pesticide market in China has exceeded 95% [1]. As one type of gram-positive bacteria [2], Bt exhibits a wide global distribution. During the formation of spores, Bt produces parasporal crystals whose main component is insecticidal crystal proteins, such as the Cry protein. The key function of the Cry protein is to interact with specific receptors on insect midgut brush border membrane vesicles (BBMVs), such as cadherin (CAD), aminopeptidase N (APN), alkaline phosphatase (ALP) and ABC transporter [1, 3]. As shown in our recent study, Cry-binding proteins are present in the midgut juice of insects and might interfere with the insecticidal process of Cry toxins. Trypsin-like serine proteases and Dorsal (a member of NF-κB transcription factors in arthropods that regulates the expression of the downstream target genes in the Toll signaling pathway [4]) were reported to be Cry1Ab1-binding proteins in the midgut juice of Plutella xylostella, and Peroxidase-C was a Cry1Ab-binding protein in Spodoptera exigua [5]. Additionally, galectin-14 was reported to compete with Cry11Aa for binding to BBMVs and ALP1 in Aedes aegypti to prevent effective binding of the toxin to receptors [6]. Hence, the mechanisms for Cry toxins against insects are believed to be much more complicated than before.

Aphididae (aphid) is one type of sucking insects of the order Hemiptera. Approximately 4,700 aphid species have been identified, of which approximately 450 damage crops [7]. Aphids suck plant sap using sharp mouthparts, and chlorophyllase and pectinase in their saliva affect plant growth and development [8, 9]. Moreover, aphids, such as peach aphid, are important viral vectors [10] and have been shown to spread more than 100 plant viruses [11]. Therefore, the harm and loss caused by viral diseases mediated by aphid-induced transmission are far greater than the damage caused by direct sucking [12].

Although chemical insecticides are still mainly used to control aphids, pathogenic microorganisms, such as viruses, fungi, bacteria, and parasitic wasps, have been considered substitutes to control aphids, due to effects on the environment and human health and the resistance of aphids to various pesticides [13–15]. Researchers planned to use Cry protein for aphid control, however, Cry protein displayed very low level of toxicity to sucking insects (Hemiptera) [16].

In this paper, we performed the assay using a typical Cry protein Cry1Ab1 to determine its effects on the most destructive aphid M. persicae, and we identified the proteins bound by Cry1Ab1 toxic core using pull-down and LC-MS/MS assays. Molecular dockings between Cry1Ab1 toxic core and its binding proteins were performed to find out the way they interact. Furthermore, Cry-binding proteins were submitted to the STRING database to analyze their functions and associated mechanisms. We then proposed a model for the inhibitory effect of Cry1Ab1 on the aphid development and reproduction, and we speculated that the Cry1A protein had the ability to disrupt aphid-mediated virus transmission. The results will result in new insights for aphid-effective Cry1A evaluation and improvement.

Experimental materials

M. persicae were reared on Brassica chinensis L., which was cultured in an artificial climate chamber (JINGHONG, Shanghai, China) with temperature of 22±2°C, relative humidity of 70-80%, and photoperiod of L:D=16:8. The Escherichia coli strain expressing the Cry1Ab1 toxic core was preserved in our lab. CNBr-activated Sepharose^TM 4B was purchased (GE Healthcare, Fairfield, CT, U.S.A.).

Preparation of the Cry1Ab1 toxic core

The expression and purification of the Cry1Ab1 toxic core were performed using the methods described by Lu [5].

Bioassay of the effects of the Cry1Ab1 toxic core on M. persicae (membrane capsule method)

The stretched 8*8 cm² parafilm was attached to one end of a cylinder (diameter of 4 cm, height of 3 cm, and wall thickness of 2 mm) with a circular cross section. Five milliliters of culture medium (containing 10, 100, 150, or 250 µg /ml Cry1Ab1) were added to the membrane, which was subsequently covered with a layer of parafilm to form a capsule.

After M. persicae were cultured for several generations in the incubator, 30 healthy 4th instars M. persicae were placed in the lower layer of each capsule, each treatment was repeated 3 times. A 6*6 cm parafilm square with 20 to 30 vent holes (transparent and breathable film) was used to seal the other end of the cylindrical bracket. The bioassay device was placed at a temperature of 18-20°C, humidity of 80%, and photoperiod of L:D=16:8 for 7 days. The aphids were gently touched with a brush at the same time every day to assess death (non-responders were considered dead), and the data were recorded for the statistical analysis.

Pull down experiment

The M. persicae homogenate was extracted from 200 healthy 3th instars M. persicae using a tissue grinder. The pull down experiment was performed using the method described by Lu [5], and the results of the separation were analyzed using polyacrylamide gel electrophoresis (SDS-PAGE), followed by silver staining.

LC-MS/MS analysis

The silver-stained SDS-PAGE gel was carefully observed, and the bands that appeared in all treatment groups, but not in the control group, were excised and sent to Beijing Huada Protein Research and Development Center (HPRC) for LC-MS/MS analysis. HPRC performed a search of the LC-MS/MS data against the uni-Aphidoidea 33385 database (52,503 sequences and 17,662,111 residues) using the MASCOT search engine (Matrix Science, London, UK). The results were submitted to the BLAST program of the National Center of Biotechnology Information (NCBI), and the accession numbers and related information of the interacting proteins were subsequently obtained.

Molecular docking and binding sites analysis

Three-dimensional (3D) structures of Cry1Ab1 toxic core and Cry-binding proteins were predicted by PHYRE2. GRAMM-X Protein-Protein Docking Web Server v.1.2.0 was used to predict the molecular docking of Cry1Ab1 toxic core and Cry-binding proteins, and then the docking results was analyzed and showed in the Discovery Studio 2.5 [6, 17]. The functional domain of Cry-binding proteins was acquired from the UniProt database.

Protein-protein interaction analysis

The STRING database integrates protein-protein interaction data from numerous predicted and known organisms, including direct (physical) interactions with specific biological functions and indirect (functional) interactions [18]. Therefore, the sequences of those proteins binding to the Cry1Ab1 toxic core were submitted to the STRING server for the analysis of functional protein interaction networks.

The Cry1Ab1 toxic core rarely kills the aphid M. persicae

The assay against M. persicae using the membrane capsule method was performed with four different concentrations of the purified Cry1Ab1 toxic core (Fig. 1). The corrected mortality rates of the Cry1Ab1 toxic core at concentrations of 10, 100, 150, and 250 µg/ml were 0.11, 0.13, 0.15 and 0.17, respectively. The calculated virulence regression equation was y=-0.0003x+0.8926, and the correlation coefficient was p=0.9857>0.95. The LC₅₀ was calculated to be 1308.6 µg/ml >> 500 µg/ml, indicating that the Cry1Ab1 toxic core rarely kills M. persicae. This result is consistent with the finding reported by Porcar, who showed that the activity of the trypsin-activated Cry1Ab toward Acyrthosiphon pisum using artificial diet bioassays was very low, with only a 25% mortality rate observed at a high concentration (500 µg/ml) [19].

GPN-loop GTPase 2, beta-actin, ATP synthase subunit alpha and an unknown and annotated protein in M. persicae were pulled down as proteins that bound to the Cry1Ab1 toxic core

The proteins that bound to the Cry1Ab1 toxic core were isolated using the pull down method, separated on SDS-PAGE gels, and subjected to silver staining (Fig. 2). The treatment group displayed an obvious band at approximately 55 kDa compared with the control. The LC-MS/MS results are shown in Table 1. The BLAST analysis revealed that the proteins that bound to the Cry1Ab1 toxic core were beta-actin (β-actin), ATP synthase subunit alpha, GPN-loop GTPase2 (Gpn2) of the GTPase protein family and an unannotated protein. Some of these proteins or their homologues have been identified as Cry-binding proteins in many insects. V-ATPase subunit A and actin were identified as CrylAc-binding proteins in the midgut of Heliothis virescens [20]; V-ATPase subunit B and actin were identified as CrylAc-binding proteins in Helicoverpa armigera [21]. Hence, ATPase and actin are two common Cry-binding proteins in insects. The fact that the proteins we identified are common Cry-binding proteins suggested that the protocol used in this paper was suitable for isolating and identifying Cry-binding proteins. Intriguingly, Gpn2, a member of the GPN family of GTPases, was first described in this paper as a Cry-binding protein. GTPase bind and hydrolyze GTP to produce GDP and inorganic phosphate, and they have various functions, including protein synthesis, cell differentiation, energy metabolism and signal transduction [22–24].

Table 1

LC-MS/MS identification for Cry1Ab1 toxic core binding proteins in M. *Persicae*
Sample	Accession number	Sore	Protein description
1	tr\|T1UMS9\|T1UMS9_APHGO	82	Beta-actin OS=Aphis gossypii
2	tr\|J9K1R5\|J9K1R5_ACYPI	31	GPN-loop GTPase 2 OS=Acyrthosiphon pisum
3	tr\|J9M0Y0\|J9M0Y0_ACYPI	29	Uncharacterized protein OS=Acyrthosiphon pisum
4	tr\|Q5XUA1\|Q5XUA1_TOXCI	28	ATP synthase subunit alpha OS=Toxoptera citricida

Cry1Ab1 toxic core bind to the functional domain of Cry-binding proteins and form different complexes

3D-Structure of Cry1Ab1 toxic core and its binding proteins including ATP synthase subunit alpha, β-actin and Gpn2 were predicted (Fig. 3). Molecular docking was performed (Fig. 4-a, b, c), and the interacting residues were showed in yellow.

For ATP synthase subunit alpha, there are three functional domains, 67-133, 190-413 and 420-545, respectively. Its interface with Cry1Ab is involved in 55E, 57-63ILGAPPK, 67-68EE, 98S, 100L, 118-119GN, 121-122KL, 128V, 130K, 142-143ED, 150D, 154-165NTIDGKGPLTSK, 251R and 278-280SDA, which located in the first and second functional domain (Fig. 4-d). There is only one functional domain in β-actin, 3-376. Its interface with Cry1Ab is involved in 40R, 60Q, 63-70RGILTLKY, 73-74EH, 158-160DGV, 180-185DLAGRD, 188-189DY, 191-192MK, 196E, 207R, 225-226EQ, 228-230MAT, 232-233AA, 264Q, 266-273SFLGMESC and 277E (Fig. 4-e). Similarly, Gpn2 has one functional domain, 8-257. Its interface with Cry1Ab is involved in 145-148SDPG, 147-148YI, 154L, 178-181AVKH, 184-185KL, 188-192NLDFY, 194-195DV, 201L, 205L and 242R (Fig. 4-f).

The interaction residues on Cry1Ab1 toxic core with ATP synthase subunit alpha were widely distributed in Domain Ⅰ, Domain Ⅱ and Domain Ⅲ, including 51-54VPGA, 114F, 117-122WEADPT, 178F, 181R and 256-258PIR in Domain Ⅰ, 349-354RIVAQL, 390Y, 392-397TSSNLP, 423R and 458R in Domain Ⅱ as well as 465-466II, 469S, 514-521NITAPLSQ, 578-580SNG and 608-610EVT in Domain Ⅲ (Fig. 5-a). The interaction residues on Cry1Ab1 toxic core with β-actin were mainly concentrated in Domain Ⅱ and Domain Ⅲ, including 292-299RSPHLMDI, 323-326ASPV, 331-337PEFTFPL, 339G, 437R and 440-442FSN in Domain Ⅱ and 479-488STNLGSGTSV, 501-507RRTSPGQ, 587S and 589-594HVFNSG in Domain Ⅲ (Fig. 5-b). Interestingly, the interaction residues on Cry1Ab1 toxic core with Gpn2 were highly similar with that of β-actin, including 289-298GSIRSPHLMD, 324-326SPV, 329-339SGPEFTFPLYG, 435-437MFR and 440-442FSN in Domain Ⅱ and 477T, 479-489STNLGSGTSVV, 501-505RRTSP and 507Q in Domain Ⅲ (Fig. 5-c). The interface of ATP synthase subunit alpha on Cry1Ab1 toxic core does not overlap with that of β-actin or Gpn2, and ATP synthase subunit alpha bind on one side and β-actin or Gpn2 on another side, indicating that ATP synthase subunit alpha, β-actin and Cry1Ab1 toxic core form a complex while ATP synthase subunit alpha, Gpn2 and Cry1Ab1 toxic core form another complex. Then, the complex 3D models were constructed and displayed (Fig. 5-d and Fig. 5-e). ATP synthase subunit alpha (yellow) and β-actin (purple) bind to Cry1Ab1 toxic core (green) on different sides (Fig. 5-d). Similarly, ATP synthase subunit alpha (yellow) and Gpn2 (blue) bind to Cry1Ab1 toxic core (green) on different sides (Fig. 5-e).

The proteins that bound to Cry1Ab1 toxic core are involved in the growth and development of aphids

The sequences of the proteins that bound to the Cry1Ab1 toxic core were submitted to the STRING server for analyses of the related pathways. Their functional distributions are shown in Fig. 6. These proteins are mainly involved in cell phagocytosis, RNA polymerase, cellular oxidative phosphorylation and other related functions. These processes or pathways play an important role in the growth and development of organisms. Phagocytosis is a highly conserved innate immune mechanism that not only eliminates foreign microbial pathogens but also digests apoptotic or necrotic cell debris from many cells. When phagocytosis occurs, foreign substances are recognized by cells, bound to the cell surface, and engulfed into the cells by phagosomes [25, 26]. RNA polymerase is involved in gene transcription and is very important for the normal expression of genes and organization of development [27]. Oxidative phosphorylation refers to the process of releasing energy as a result of the oxidation of organic matter in the organism and drives the synthesis of ATP, which is necessary for sustaining the synthesis of chemicals that maintain metabolism and cell structure, and for the transport of the ions and molecules that maintain the intracellular environment [28].

Although the Cry toxin protein possesses specific insecticidal activity against a variety of agricultural and forestry pests, aphids are not sensitive to it [16]. Therefore, the study aiming to uncover the reasons for this insensitivity will give us valuable implications for evaluating and improving its activity against the aphid. We confirmed that Cry1Ab1 toxic core exerted very low ability for killing M. persicae, then we identified the proteins that bind to the Cry1Ab1 toxic core in M. persicae, and analyzed the docking interface between Cry1Ab1 and its three binding proteins, ATP synthase subunit alpha, beta-actin and Gpn2, performed STRING database searching to find their related pathways, all those suggesting that they formed protein complex to disturb the growth and development of the aphid.

ATPase, which is known as the “pH meter” of the cell, regulates pH to modulate enzymatic activity, thereby affecting the metabolic rate of the cell. V-ATPase on the membrane also functions as a receptor to regulate the pH-mediated endocytosis and exocytosis in cells [29, 30]. According to Whyard et al, the consumption of the V-ATPase dsRNA caused the death of Drosophila melanogaster, Tribolium castaneum, A. pisum and Manduca sexta [31]. ATP synthase subunit alpha consists of a soluble domain at the N-terminus in the cytoplasm and a transmembrane region containing 9 α-helices at the C-terminus. Several amino acid residues located at the C-terminus participate in proton transport and the formation of H⁺ channels [32]. According to the chemical permeation theory, V-ATPase transports protons, and ATP is synthesized using the H⁺ potential during the process of oxidative phosphorylation. Based on the analysis described above, ATP synthase subunit alpha is presumed to participate in the proton transport required for ATP synthesis mediated by oxidative phosphorylation. Meanwhile, Docking interface analysis indicated that Cry1Ab1 toxic core bound with the functional domain of ATP synthase subunit alpha, Therefore, the interaction between Cry1Ab1 toxic core and ATP synthase subunit alpha might interfere with oxidative phosphorylation, affecting the normal energy supply in the cells and thereby disrupting the metabolic reactions in the cells and even inhibiting the growth and development of aphids.

Actin has been identified as one of the Cry-binding proteins in Mythimna separate [33], H. virescens [20], H. armigera [21], A. aegypti [34]. Vega-Cabrera also observed an interaction between Cry11Aa toxin and actin on the cytoplasmic side of the membrane after toxin penetration [35]. In the present study, β-actin in M. persicae bound to the Cry1Ab1 toxic core. β-actin is involved in a wide range of cellular activities, including the transport of intracellular substances, cellular shape associated with cell movement, endocytosis, cytokinesis and cell adhesion [36–39]. Notably, Cry toxins belong to the pore-forming toxin family (PFT), which is the most widespread group of toxins produced by bacteria [40]. The interaction of PFT and actin not only enhances actin polymerization in vitro [41] but indirectly promotes toxin oligomerization and endocytosis [42]. Therefore, we speculated that β-actin mediated the endocytosis of Cry1Ab1 located in cell membrane, and β-actin functioned as a cytoskeletal protein that binds to Cry1Ab1 and transports it to an intracellular site as the cytoplasm flows. β-actin is tightly integrated with RNA polymerase III (RNAP III) by interacting with RPABC3 (RPC62), RPABC2 (RPB6) and RPABC3 (RPB8) on RNAP III [43]. Interestingly, RPABC2 and RPABC3, which are common subunits of RNAP Ⅰ, RNAP Ⅱ and RNAP Ⅲ [44], were detected to be adjacent to each other in the crystal structure of RNAP Ⅱ [45], and β-actin has been shown to interact with RNAP Ⅰ [46]. Based on the analysis described above, we concluded that β-actin firmly binds to three RNAP by interacting with adjacent RPABC2, RPABC3 and other subunits, indicating that β-actin was quite important in the formation and function of RNAP. Once Cry1Ab1 bound to β-actin, it might hinder the normal assembly of RNAP, affecting the normal transcription and expression of genes, and then blocking the development and metabolism of M. persicae. Captivatingly, actin has also been reported to function as a receptor of beet western yellows virus particles in aphids, and probably played a role in the transcytosis and transmission of the virus [47]. Using a genomic analysis method, Tamborindeguy found that several actin proteins are potentially involved in the transcytosis of viruses in A. pisum [48]. Thus, after digestion by M. persicae, the binding of Cry1Ab1 to actin likely disturbs the interaction of actin and the virus, affecting virus transmission mediated by M. persicae.

The GPN family, including Gpn1, Gpn2 and Gpn3, is known for its invariant glycine–proline–asparagine structural loop [49]. Gpn2 is required for the proper localization of RNAPII and RNAPIII. As shown in the study by Zeng et al, Gpn2 might interact with RNA polymerases [27], which have important roles in gene expression, cell fate determination and tissue development by catalyzing the synthesis of mRNA and non-coding RNA in eukaryotes [50–52]. Zeng et al confirmed that Gpn2 and Rba50 are directly involved in the assembly of the Rpb3 subcomplex that bridges the Rpb1 and Rpb2 subcomplexes to produce the core of RNAPII, and even subsequent biogenesis of RNAPII [27]. Because β-actin might also bind tightly to RNAP and be involved in the formation and function of RNAP, the interaction of Cry1Ab1 with Gpn2 or β-actin would probably significantly disrupt the assembly and function of RNAP, as well as DNA transcription and expression, subsequently resulting in some damage to the metabolism and development of M. persicae.

From the above analysis, Cry1Ab1 toxic core interacted with Cry-binding protein including ATP synthase subunit alpha, β-actin and Gpn2, it would eventually affect the metabolism and development of M. persicae. Considering the pull down results and docking analysis, it is likely that Cry1Ab1 toxic core bind with ATP synthase subunit alpha and β-actin or ATP synthase subunit alpha and Gpn2 to form protein complex, and the protein complex probably covered the functional domain of three Cry-binding proteins and dragged the ATP synthase subunit alpha, β-actin or Gpn2 to non-functional area of certain Cry-binding protein, thereby potentially interrupting the normal energy supply, blocking the normal transcription and expression of genes and would eventually affect the metabolism and development of M. persicae.

Zhang et al identified Cry1Ac/Ab in unfed Bt coccinellid Propylaea japonica neonates whose parents fed on aphids reared on Bt cotton [53], suggesting that Cry1Ac/Ab enters the organelles and cell through parental transfer, and Cry1Ab1 protein was unable to be degraded and activated due to the neutral environment in the midgut of aphid [54]. This study proposed a hypothetical model for the molecular mechanism underlying the effect of Cry1Ab1 on aphids (Fig. 7). From the hypothetical model, Cry1Ab1 protein is unable to kill the aphid, but potentially affects development and reproduction. Some studies indicated an effect of Cry proteins on the growth and development of sucking insects. According to Tan et al, the non-target pest Sogatella furcifera (Homoptera: Delphacidae) exhibits a significantly longer developmental duration than the controls after feeding on insect-resistant transgenic indica rice carrying the cry1Ab gene [55]. Azimi reported a delay in the progression of Bemisia tabaci (Hemiptera) fed on Bt cotton expressing the Cry1Ab protein to adulthood later and a lower level of fertility than insects fed on non-Bt cotton [56]. Additionally, Porcar announced that after ingesting Cry1Ab, the growth rates of surviving A. pisum were markedly reduced compared to the control group [19]. Because the capsule method was a short-term bioassay that did not reflect the long-term effects of the Cry toxin on aphids, the Cry1Ab1 toxic core rarely directly kills aphids, but it might affect the development of aphids and the reproduction of populations, similar to Cry1Ac (86.17% of the amino acid sequence is the same as Cry1Ab1), which is less toxic to M. persicae but inhibits the population growth rate [57]. The explanations for the inhibitory effects, however, remain unknown. Our study provided clues for the molecular mechanism of inhibition, namely, the proteins involved in cellular metabolic processes in M. persicae are bound by the Cry1Ab1 toxic core.

Based on the results reported by Pascale et al, actin is involved in the epithelial transcytosis of virus particles in the aphid vector [47]. This finding and the observation that actin is a common Cry-binding protein in our study and other studies prompted us to speculate that the Cry protein disrupt virus transmission mediated by aphids, providing a new strategy for controlling viral diseases mediated by aphids. Additionally, APN and translation elongation factor, which have been identified as other Cry-binding proteins in the aphid (unpublished data from our lab), were also proven to be involved in the interaction with the virus in aphids [58, 59]. APN is the receptor for numerous coronaviruses, such as porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV) [60]. Linz et al recently confirmed that the coat protein (CP) of pea enation mosaic virus (PEMV) binds to its receptor APN in the gut of the pea aphid [58]. The interaction between elongation factor and viral RNA-dependent RNA polymerase (RdRp) or viral genomic RNA promotes virus replication and transmission [59]. Thus, some aphid proteins are simultaneously bound by viral proteins and Cry proteins, further supporting our hypothesis that the Cry protein disrupts virus transmission mediated by aphids.

Last but not the least, based on the long-term feeding effect of Cry protein expressed in plant on aphid population, and the possibility that Cry protein disrupt virus transmission, when we screen anti-aphid Cry proteins, we should not only examine the short-term killing ability but also focus on the long-term effects on the growth and reproduction and virus transmission by aphids.

Author Contribution JL and Z.B.W: designed and performed the experiment; JL: wrote the manuscript; JL and Z.X.D: reviewed the manuscript; L.Y: revised and approved the manuscript.

Funding This study was supported by grants from the National Key Research and Development Program of China (Grant No. 2017YFD0201201), NNSFC (National Natural Science Foundation of China, Grant No. 31772227).

Availability of Data and Materials Not applicable.

Ethics Approval and Consent to Participate Not applicable.

Consent to Participate Not applicable.

Consent to Publish Not applicable.

Conflict of Interest Statement The authors declare that they have no competing interests.

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Indications for the Potential of Cry1Ab1 for Inhibiting Aphid Development and Virus Transmission

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Abstract

Figures

Introduction

Materials And Methods

Experimental materials

Preparation of the Cry1Ab1 toxic core

Pull down experiment

LC-MS/MS analysis

Molecular docking and binding sites analysis

Protein-protein interaction analysis

Results

Cry1Ab1 toxic core bind to the functional domain of Cry-binding proteins and form different complexes

Conclusion And Discussion

Declarations

REFERENCES

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