Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta

Onofre, Janette; Gaytán, Meztlli O.; Peña-Cardeña, Arlen; García‐Gómez, Blanca I.; Pacheco, Sabino; Gómez, Isabel; Bravo, Alejandra; Soberón, Mário

doi:10.1016/j.peptides.2017.01.006

Cited by 14 publications

(7 citation statements)

References 27 publications

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“…MALDI-TOF/TOF analyses further confirmed that Cry interacting protein (~110 kDa) in A. janata BBMVs is aminopeptidase N. Colocalization studies substantiate the presence of APN and Cry toxin in close proximity in apical brush border membrane of midgut epithelium. The results obtained in present study corroborate well with earlier reports where brush border membrane region was shown to interact and accumulate toxins in intoxicated larvae (Bravo et al, 2007; Yi et al, 1996; Valaitis, 2011; Onofre et al, 2017). Reported molecular weights of APNs in different insects ranged between 100-150 kDa.…”

Section: Discussionsupporting

confidence: 92%

Midgut aminopeptidase N expression profile in Castor semilooper during sublethal Cry toxin exposure

Chauhan

Dhania

Lokya

et al. 2019

Preprint

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31Midgut of lepidopteran larvae is a multifunctional tissue, which performs roles in digestion, 32 absorption, immunity; transmission of pathogens and interaction with ingested various 33 molecules. The proteins localized at the inner apical brush border membrane are primarily 34 digestive proteases but some of them like aminopeptidase N, alkaline phosphatase, cadherins, 35 ABC transporter C2 etc. interact with Crystal (Cry) toxins produced by Bacillus thuringiensis 36 (Bt). In the present study aminopeptidase N (APN) was characterized as Cry toxin interacting 37 protein in larval midgut of castor semilooper, Achaea janata. Transcriptomic and proteomic 38 analyses revealed the presence of multiple isoforms of APNs (APN1, 2, 4, 6 and 9) which 39 have less than 40% sequence similarity but show the presence of characteristic "GAMENEG" 40 and zinc-binding motifs. Feeding of sublethal dose of Cry toxin caused differential 41 expression of various APN isoform. Further, 6 th generation Cry toxin exposed larvae showed 42 reduced expression of APN2. This report suggests that A. janata larvae exploit altered 43 expression of APNs to overcome the deleterious effects of Cry toxicity, which might 44 facilitate toxin tolerance in long run. 45 46 47 93 2019).94 Bt-based biopesticides are currently being used as an alternative to chemical insecticides for 95 the effective management of economically important Lepidopteran pests either Bt based 96 formulations or Bt transgenic plants. However, development of resistance against Cry toxin 97based control strategies in insect populations is a great concern and needs a careful evaluation 98 (Tabashniket al., 2013). Reports related to the development of resistance in laboratory based 99 as well as field collected populations shown to mediated by multiple mechanisms (Pardo-100 Lopez et al., 2013; de Bortoli and Jurat-Fuentes, 2019). In certain species, it was due to the 101 alteration in Cry toxin activation by gut proteases (Keller et al., 1996; Oppert et al., 1997; Li 102 et al., 2004), sequestration of Cry toxins by glycolipid moieties (Ma et al., 2005) and an 103 elevated immune response (Hernández-Martínez et al., 2010) were additional mechanisms 104 which promoted the development of Cry resistance. Mutation in APN receptor proteins was 105 shown to be associated with reduced interaction of Cry toxin with larval midgut (Zhang et al., 106 2009). Tiewsiri and Wang (2011) correlated toxin resistance with a reduced and differential 107 expression of various APN isoforms in T. ni. Recently Sun et al., (2019) demonstrated that 108 knockdown of APN isoforms 6 and 8 decreases the susceptibility of Chilo suppressalis larvae 109 towards Cry toxins. 110 In the present study, an attempt was made to characterize the aminopeptidase N as Cry toxin 111 receptor/binding protein in the larval midgut of Achaea janata, commonly known as castor 112 semilooper a serious lepidopteran pest of castor plantation in India. Further, various isoform 113 of APNs were identified and their expression profil...

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Section: Discussionsupporting

confidence: 92%

Midgut aminopeptidase N expression profile in Castor semilooper during sublethal Cry toxin exposure

Chauhan

Dhania

Lokya

et al. 2019

Preprint

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“…However, for a better understanding of the interactions between Cry toxins and APNs in Lepidoptera, it will be important to perform the functional characterization of some APN sequences found in this study. Successful attempts to identify the specificity of APN receptors to certain Cry toxins have been performed in the past, laying the basis for their biotechnological application [73][74][75].…”

Section: Plos Onementioning

confidence: 99%

Comparative gut transcriptome analysis of Diatraea saccharalis in response to the dietary source

et al. 2020

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The sugarcane borer (Diatraea saccharalis, Fabricius, 1794) is a devastating pest that causes millions of dollars of losses each year to sugarcane producers by reducing sugar and ethanol yields. The control of this pest is difficult due to its endophytic behavior and rapid development. Pest management through biotechnological approaches has emerged in recent years as an alternative to currently applied methods. Genetic information about the target pests is often required to perform biotechnology-based management. The genomic and transcriptomic data for D. saccharalis are very limited. Herein, we report a tissue-specific transcriptome of D. saccharalis larvae and a differential expression analysis highlighting the physiological characteristics of this pest in response to two different diets: sugarcane and an artificial diet. Sequencing was performed on the Illumina HiSeq 2000 platform, and a de novo assembly was generated. A total of 27,626 protein-coding unigenes were identified, among which 1,934 sequences were differentially expressed between treatments. Processes such as defence, digestion, detoxification, signaling, and transport were highly represented among the differentially expressed genes (DEGs). Furthermore, seven aminopeptidase genes were identified as candidates to encode receptors of Cry proteins, which are toxins of Bacillus thuringiensis used to control lepidopteran pests. Since plantinsect interactions have produced a considerable number of adaptive responses in hosts and herbivorous insects, the success of phytophagous insects relies on their ability to overcome challenges such as the response to plant defences and the intake of nutrients. In this study, we identified metabolic pathways and specific genes involved in these processes. Thus, our data strongly contribute to the knowledge advancement of insect transcripts, which can be a source of target genes for pest management.

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“…Therefore, knowledge of the specific receptor utilization step in the overall mechanism of action (MOA) of pore-forming insecticidal proteins is a key component in deploying new IP maize products for increased durability (31–34). There are several methods used to study the receptor utilization of insecticidal proteins, including ligand blots (35, 36), in vitro binding experiments with labeled insecticidal proteins (30) and isolated insect gut brush border membrane vesicle (BBMV) preparations (37), pulldown experiments using immobilized or immunoprecipitated insecticidal proteins (38), insect cell-based assays using cloned insect receptor genes (39, 40), the disabled insecticidal protein (DIP) assay (41), and experiments with resistant insect colonies (42–45). Here, we report the development of two new modified insecticidal proteins for use against FAW, Cry1B.868 and Cry1Da_7, with enhanced specific activity against FAW and corn earworms (CEW), Helicoverpa zea (Boddie), respectively, and our comprehensive assessment of their FAW receptor preferences based on available resistant colonies, DIP assays, and cell-based receptor screens.…”

Section: Introductionmentioning

confidence: 99%

Bacillus thuringiensis Cry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms, Spodoptera frugiperda (J.E. Smith)

Wang

et al. 2019

Appl Environ Microbiol

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Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest. IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.

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Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta

Cited by 14 publications

References 27 publications

Midgut aminopeptidase N expression profile in Castor semilooper during sublethal Cry toxin exposure

Midgut aminopeptidase N expression profile in Castor semilooper during sublethal Cry toxin exposure

Comparative gut transcriptome analysis of Diatraea saccharalis in response to the dietary source

Bacillus thuringiensis Cry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms, Spodoptera frugiperda (J.E. Smith)

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