Alkaline phosphatase 2 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea

Abstract

Although membrane‐bound alkaline phosphatases (ALPs) have been proposed as a receptor for Cry1Ac in a few lepidopteran species, their potential functions as a Cry2Ab receptor are yet to be verified. To determine if ALP2 also serves as a receptor for Cry1Ac and even for Cry2Ab in Helicoverpa zea, we measured the potency of activated Cry1Ac and Cry2Ab against midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) expressing H. zea ALP2 (HzALP2) or transfected with HzALP2 double‐stranded RNA (dsRNA). Relative to the control cells, the three cell lines expressing HzALP2 were more susceptible to Cry1Ac but there was no difference for Cry2Ab. By contrast, the two H. zea cell lines transfected with HzALP2 dsRNA were resistant to Cry1Ac while kept susceptible to Cry2Ab. Furthermore, RNA interference knockdown of HzALP2 in H. zea larvae enhanced larval survival on Cry1Ac‐containing diets. These findings indicate that HzALP2 functions as a receptor of Cry1Ac but not Cry2Ab.

Polycalin is involved in the toxicity and resistance to Cry1Ac toxin in Helicoverpa armigera (Hübner)

“…Meanwhile, the up-or downregulation of these receptors have been reported to be involved in the insect resistance to Cry toxins (Atsumi et al, 2012;Baxter et al, 2011;Chen et al, 2015;Gahan, Pauchet, Vogel, & Heckel, 2010;Jurat-Fuentes et al, 2011;Tiewsiri & Wang, 2011;Xiao et al, 2014;Xu, Yu, & Wu, 2005;Zhang et al, 2009). Along with the further research, more new receptor proteins, new genes, and resistance pathways are being reported to be involved in the toxicity of Bt and the resistance mechanisms of insects (Heckel, 2012;Wei, Zhang, Liang, & Li, 2018).…”

Suppression of Calcineurin Enhances the Toxicity of Cry1Ac to Helicoverpa armigera

“…Cell mortality was calculated with the Abbott formula (Abbott, 1925) as the description in Wei et al (2016Wei et al ( , 2019b). Significant differences in cell mortalities, relative expression level, and enzyme activities of HaCAN between CAN-GFP-pIEx (dsCAN) and GFP-pIEx (dsEGFP) were compared using Student's t-test (DPSSOFT: DPS7.05).…”

“…Helicoverpa zea midgut cell line RP-HzGUT-AW1 (MG) was generously provided by Dr. Xianchun Li (University of Arizona, Tucson, AZ, United States) (Goodman et al, 2004). Spodoptera frugiperda cell line (Sf9) and MG cell line were routinely maintained at a constant temperature incubator, and the culture mediums were prepared as the description in Wei et al (2016Wei et al ( , 2018aWei et al ( , 2019b.…”

“…Empty pIEx-RFP (or DsEGFP) vector was used as control. Five hours after transfection, transfection mixture was removed and replaced by the newly supplemented medium (according to the description in Wei et al, 2016Wei et al, , 2018aWei et al, , 2019b. Sixty-four hours after cell maintenance at 28 • C, the cells that transfected pIEx-RFP-CAN or pIEx-RFP were observed under the fluorescence microscope (Zeiss, LSM780) to check the expression of proteins.…”

“…Sixty-four hours after cell maintenance at 28 • C, the cells that transfected pIEx-RFP-CAN or pIEx-RFP were observed under the fluorescence microscope (Zeiss, LSM780) to check the expression of proteins. After 64 h post-transfection, the cells were also collected and the concentration of cells was measured with hemocytometer by using trypan blue; then the treated cells were reseeded in a 96-well micro-plate (costar) for 100 µL of cells (10,000 cells), allowing cells to attach about 2 h. We tested the cytotoxicity of the treated cells by using Cry1Ac toxin (100 µg/mL for expressing HaCAN and 50 µg/mL for knockdown of HaCAN) for 4 h; finally, the cell mortality was calculated under the inverted microscope (OPTIKA IM-5) (Wei et al, 2016(Wei et al, , 2018a(Wei et al, , 2019b. The remaining treated cells of each independent transfection were collected, centrifuged at 1000 × g for 10 min, washed with an equal volume of cold PBS buffer for three times, and then stored them at −80 • C for subsequent RNA or protein extraction to make sure the successful transfection.…”