APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea.

Jizhen Wei,Yuyuan Guo,Min Zhang,Gemei Liang,Xinzhi Ni,Kongming Wu,Xianchun Li

doi:10.1038/srep19179

Abstract

Lepidopteran midgut aminopeptidases N (APNs) are phylogenetically divided into eight clusters, designated as APN1–8. Although APN1 has been implicated as one of the receptors for Cry1Ac in several species, its potential role in the mode of action of Cry2Ab has not been functionally determined so far. To test whether APN1 also acts as one of the receptors for Cry1Ac in Helicoverpa zea and even for Cry2Ab in this species, we conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by RNAi (RNA interference) silencing of the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA (dsRNA). Heterologous expression of HzAPN1 significantly increased the susceptibility of the three cell lines to Cry1Ac, but had no effects on their susceptibility to Cry2Ab. Knocking down of the endogenous APN1 made the three cell lines resistant to Cry1Ac, but didn’t change cell lines susceptibility to Cry2Ab. The findings from this study demonstrate that HzAPN1 is a functional receptor of Cry1Ac, but not Cry2Ab.

Highlights

Aminopeptidases N (APNs) are a class of broad-specificity metalloaminopeptidases that sequentially and preferentially remove neutral amino acids from the N-terminus of a range of peptides in microorganisms, plants and animals[1]
Conflicting evidence exists for APN2, because 1) it can bind to Cry1Ac in Helicoverpa armigera[15], but not in Lymantria dispar[16]; and 2) it is not linked to Cry1Ac resistance in Plutella xylostella[17]
We conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by knocking down the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA

Summary

Introduction

Aminopeptidases N (APNs) are a class of broad-specificity metalloaminopeptidases that sequentially and preferentially remove neutral amino acids from the N-terminus of a range of peptides in microorganisms, plants and animals[1]. Presence of cross-resistance between Cry1Ac and Cry2Ab27–29 and detection of a Cry1Ac- and Cry2Ab-binding protein with a similar size (120 ~ 130 kDa) of APN1 in the midgut and fat body cell lines of H. zea by ligand blot suggest a possibility of APN1 as a shared receptor for Cry2Ab and Cry1Ac (Wei & Li, unpublished manuscript). To test this hypothesis, we conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by knocking down the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA (dsRNA). The data demonstrate that APN1 is a receptor for Cry1Ac, but not for Cry2Ab

Methods

Results

Conclusion