Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system

Wang, Jing; Zhang, Haonan; Wang, Huidong; Zhao, Shan; Zuo, Yayun; Yang, Yihua; Wu, Yi

doi:10.1016/j.ibmb.2016.06.008

Cited by 128 publications

(109 citation statements)

References 59 publications

(29 reference statements)

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“…We synthesized two sgRNAs, named BdWhite‐g1 and BdWhite‐g2 , which are located in exons 1 and 3 respectively, and we injected a mixture of Cas9 protein and sgRNA, transcribed in vitro , into fresh eggs that resulted in highly efficient editing of the BdWhite locus. We observed a 100% editing frequency in the surviving flies of the BdWhite‐g2 ‐injected generation; this 100% germline transmission rate was higher than reported for other insects (Wang et al ., ; Xue et al ., ; Chen et al ., ), including B. dorsalis (Zhao et al ., ; Zheng et al ., ; Sim et al ., ). The high mutation efficiency may be due to the high concentration of sgRNA (500 ng/μl) used in this study, and rapid injection of the embryos following oviposition (within 1 h) and the injection of cas9 protein into embryos, rather than cas9 mRNA or plasmid (Zhao et al ., ; Zheng et al ., ).…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9‐mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis

Bai

Zeng

et al. 2019

Insect Molecular Biology

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The intensely studied white gene is widely used as a genetic marker in Drosophila melanogaster. Here, we cloned and characterized the white gene in an important pest of the fruit industry, Bactrocera dorsalis, to understand its functional role in pigmentation. We obtained BdWhite knockout strains, based on the wild‐type strain, using the CRISPR/Cas9 genome editing system, and found that mutants lost pigmentation in the compound eye and their black head spots. We then examined differences in the expression levels of genes associated with melanin pigmentation between mutants and the wild‐type strain using quantitative reverse transcription PCR. We found that transcription levels of the Bd‐yellow1 were lower in the head of mutants than in the wild‐type strain, and there were no significant differences in expression of the other six genes between mutants and the wild type. Since yellow is critical for melanin biosynthesis (Heinze et al., Scientific Reports. 2017;7:4582), the lower levels of expression of Bd‐yellow1 in mutants led to reduced dark pigmentation in head spots. Our results provide the first evidence, to our knowledge, that white may play a functional role in cuticle pigmentation by affecting the expression of yellow.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9‐mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis

Bai

Zeng

et al. 2019

Insect Molecular Biology

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“…One of the major factors in evaluating efficiency of gene editing techniques is the mutation frequency. In our study, the G 0 chimera mutation frequency for the SeP‐gp gene was 33.3%, which is in agreement with some reports in the silkworm (Ma et al ., ; Wei et al ., ) but lower than the 55% cadherin gene (HaCad) mutagenesis in H. armigera (Wang et al ., ), the 87.5% pheromone binding protein 3 gene ( SlitPBP3 ) gene mutagenesis in Spodoptera litura (Zhu et al ., ) or the 95% mutagenesis achieved for the oily skin phenotype gene ( BmBLOS2 ) gene in Bombyx mori (Wang et al ., ). This discrepancy may be explained by differences in target gene selection, single guide RNA (sgRNA) or Cas9 dose, or forms of Cas9 injected (mRNA, Cas9 in plasmid or Cas9 protein) (Zhu et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Knockout of a P‐glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua

Zuo

Huang

Wang

et al. 2017

Insect Molecular Biology

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P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) geneediting technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, betacypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua.

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“…The insect midgut membrane protein CAD and the highly abundant glycosyl phosphatidylinositol (GPI)-anchored APN and ALP proteins have been characterized as functional binding receptors for Cry1A toxins in several lepidopteran insects28414243. However, limited studies have focused on defining the role of the insect membrane proteins CAD, APN and ALP in relation to Cry2A toxins to date4445.…”

Section: Discussionmentioning

confidence: 99%

Functional roles of cadherin, aminopeptidase-N and alkaline phosphatase from Helicoverpa armigera (Hübner) in the action mechanism of Bacillus thuringiensis Cry2Aa

Zhao

Yuan

Wei

et al. 2017

Sci Rep

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A pyramid strategy combining the Cry1A and Cry2A toxins in Bt crops has been widely used throughout the world to delay pest adaption to transgenic crops and broaden the insecticidal spectrum. Midgut membrane-bound cadherin (CAD), aminopeptidase-N (APN) and alkaline phosphatase (ALP) are important for Cry1A toxicity in some lepidopteran larvae, but the proteins that bind Cry2A in the midgut of target insects and their role in the Cry2A mechanism of action are still unclear. In this study, we found that heterologously expressed CAD, APN4 and ALP2 peptides from the midgut of Helicoverpa armigera could bind to the Cry2Aa toxin with a high affinity. Additionally, the efficiency of Cry2Aa insecticidal activity against H. armigera larvae was obviously reduced after the genes encoding these proteins were silenced with specific siRNAs: CAD- and ALP2-silenced larvae showed significantly similar reductions in mortality due to the Cry2Aa toxin (41.67% and 43.06%, respectively), whereas a larger reduction in mortality was observed in APN4-silenced larvae (61.11%) than in controls. These results suggest that CAD, APN4 and ALP2 are involved in the mechanism of action of Cry2Aa in H. armigera and may play important functional roles in the toxicity of the Cry2Aa toxin.

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Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system

Cited by 128 publications

References 59 publications

CRISPR/Cas9‐mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis

CRISPR/Cas9‐mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis

Knockout of a P‐glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua

Functional roles of cadherin, aminopeptidase-N and alkaline phosphatase from Helicoverpa armigera (Hübner) in the action mechanism of Bacillus thuringiensis Cry2Aa

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