ATP-Binding Cassette Subfamily a Member 2 Is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, But Not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins

Abstract

Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239T cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1Ca, Cry1Da, Cry1Fa or Cry9Aa toxins.

CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab

“…These results imply that mutations in PgABCA2 can cause resistance to Cry2Ab, which is consistent with previous results indicating that pink bollworm resistance to Cry2Ab is genetically linked with recessive mutations in PgABCA2 in two lab-selected strains from Arizona (Bt4-R2 and BX-R) and associated with mutations in PgABCA2 in field-selected populations from India 29 , 30 . The results with pink bollworm also correspond with evidence that mutations in ABCA2 genes introduced by CRISPR or TALENs caused resistance to Cry2Ab in H. armigera, T. ni, and B. mori 22 – 24 and that naturally occurring deletions at this locus were associated with resistance to Cry2Ab in H. armigera and H. punctigera 21 .…”

“…Resistance to Cry2Ab in the lepidopteran pests Helicoverpa armigera and Helicoverpa punctigera is associated with reduced binding of Cry2Ab to larval midgut membranes and is linked with autosomal recessive mutations in the ABC transporter gene ABCA2 20 , 21 . Furthermore, knockout or removal of portions of the gene encoding ABCA2 using CRISPR/Cas9 or TALENs caused resistance to Cry2Ab in H. armigera, Trichoplusia ni, and Bombyx mori 22 – 24 .…”

The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture

“…After mapping Cry2Ab resistance in T. ni to ABCA2, where a transib mobile element was found to disrupt the gene, either ABCA1 or ABCA2 were knocked out in a susceptible strain and only ABCA2 was found to affect Cry2Ab tolerance [ 69 ]. Knockouts of the ABCA2 gene in B. mori (using the TALEN technique) conferred Cry2A resistance on larvae, and heterologous expression of ABCA2 in HEK239 cells confirmed the absence of cross-resistance to Cry1A, Cry1Ca, Cry1Da, Cry1Fa, and Cry9Aa toxins [ 76 ].…”

Construction and analysis of the protein–protein interaction network for the detoxification enzymes of the silkworm, Bombyx mori

“…In insects, there are mainly three enzymes involved in detoxification: cytochromes P450 (CYPs), glutathione S-transferases (GSTs), and carboxylesterase (CarEs) (Hatfield et al, 2016;Montella et al, 2012;Rane et al, 2019;Schuler, 2011;. In addition, UDP-glycosyltransferases (UGTs) and ATP-binding cassette (ABC) transporters are also involved in the metabolism of toxic substances (Bock, 2016;Heidel-Fischer & Vogel, 2015;Li et al, 2020;Wu et al, 2019).…”