Abstract
The fall webworm, Hyphantria cunea (Drury) is a major invasive pest in China. Aminopeptidase N (APN) isoforms in lepidopteran larvae midguts are known for their involvement in the mode of action of insecticidal crystal (Cry) proteins from Bacillus thuringiensis. In the present work, we identified a putative Cry1Ab toxin-binding protein, an APN isoform designated HcAPN3, in the midgut of H. cunea by ligand blot and mass spectrometry. HcAPN3 was highly expressed throughout all larval developmental stages and was abundant in the midgut and hindgut tissues. HcAPN3 was down-regulated at 6 h, then was up-regulated significantly at 12 h and 24 h after Cry1Ab toxin treatment. We expressed HcAPN3 in insect cells and detected its interaction with Cry1Ab toxin by ligand blot assays. Furthermore, RNA interference (RNAi) against HcAPN3 using oral delivery and injection of double-stranded RNA (dsRNA) resulted in a 61–66% decrease in transcript level. Down-regulating of the expression of HcAPN3 was closely associated with reduced susceptibility of H. cunea to Cry1Ab. In addition, the HcAPN3E fragment peptide expressed in Escherichia coli enhanced Cry1Ab toxicity against H. cunea larvae. This work represents the first evidence to suggest that an APN in H. cunea is a putative binding protein involved in Cry1Ab susceptibility.
Highlights
The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae) is a severe economic pest that originated in North America [1]
Aminopeptidase N (APN) are involved in several functions in a wide range of species; in the lepidopteran larval midgut they play an important role in protein digestion, co-operating with endopeptidases and carboxypeptidases to digest proteins derived from the diet [10]
FigFuigreur1e. 1S.DSSD-PS-APGAEGE(1(01%0%) a) naanlaylysissisoof fCCryry11AAbb3355eexxpprreessed in B. thuurriinnggiieennssisisHHDD7733− −strsatirnaiannadntdhethe actaivcFtaiivtgeaudtreeCd1rC.ySr1yDA1SbA-P3bA53G5toEtxoi(xn1i0nd%dig)igeasnetsaetledydsbisbyyotfrtrCyyprpyss1iinAn.b
Summary
The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae) is a severe economic pest that originated in North America [1]. Aminopeptidase N (APN) family is composed of a class of zinc metalloproteinases that preferentially cleave single neutral amino acids from the N-terminus of polypeptides [7]. They are distributed widely in the plant and animal kingdoms and highly expressed in the brush border membranes of the alimentary tract in lepidopteran larvae [8,9]. Each lepidopteran species shows no more than one sequence for each cluster with the exception of Class 3, which includes more isoforms in several species [12,14]
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