RNAi-mediated knockdown of <i>AjAPN1</i> transcript and its encoded protein.

<p>Third instar larvae were intrahemocoelically injected with target and control gene siRNA duplexes at dose of 5 µg/100 mg body weight followed by analyses of target gene/protein expression level at different time points. Observations obtained at 66 h post-injection are represented. Values represented are mean±standard deviation of three independent experiments (n = 3). Significance between groups was tested by One-Way ANOVA followed by Student-Newman-Keuls’ (SNK) test using SigmaPlot 11.0 software. *indicate statistical significance (P<0.05). Control (Cont): double-stranded <i>GFP</i> siRNA injected and Experimental (Expt): double-stranded <i>AjAPN1</i> siRNA injected insects. (A) Real-time quantitative PCR analysis. 18S rRNA was used as an internal endogenous control. Note that the fold decrease in <i>AjAPN1</i> transcript level in fat body and Malpighian tubule was 1.9 and 2.8 respectively. Semi-quantitative analysis is represented by the gel images. Here, <i>β</i>-actin gene was used as an internal endogenous control (lower panel). (B) Western blot analysis. Note the substantial reduction in the 113 kDa AjAPN1 protein band of fat body and Malpighian tubule of the target gene siRNA injected insects. β-actin expression was used as an internal endogenous control (lower panel). (C) APN activity analysis. Note the significant decrease in the APN activity level of fat body and Malpighian tubule of the target gene siRNA injected insects. Fb: fat body, Mt: Malpighian tubule and Sg: salivary gland.</p

Analyses of Cry1Aa toxin interaction with AjAPN1.

<p>(A) Comparison of Cry1Aa toxin binding region of <i>B. mori</i> midgut APN (BmAPN) and <i>P. xylostella</i> midgut APN (PxAPN) with AjAPN1. (B) 3D structures of AjAPN1 and <i>B. mori</i> midgut APN. The secondary structure of the Cry1Aa toxin binding region located in domain I is highlighted in yellow. (C) <i>In vitro</i> Cry1Aa toxin binding analysis. Note the binding of Cry1Aa toxin to a 113 kDa membrane protein of Malpighian tubule of <i>A. janata</i>. Blot showing binding of Cry1Aa toxin to the 113 kDa protein of <i>B. mori</i> midgut BBMV act as a control. Mt: Malpighian tubule, Gt: gut, AjMtAPN: <i>A. janata</i> Malpighian tubule APN, AjFbAPN: <i>A. janata</i> fat body APN and AjSgAPN: <i>A. janata</i> salivary gland APN.</p

Characteristics of the templates.

<p>Characteristics of the templates.</p

Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest <i>Achaea janata</i>

<div><p>Insect midgut membrane-anchored aminopeptidases N <b>(</b>APNs) are Zn⁺⁺ dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific <i>in vitro</i> interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of <i>Achaea janata</i>. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of <i>A. janata</i> showed high structural similarity to the Cry1Aa toxin binding midgut APN of <i>Bombyx mori</i>, especially in the toxin binding region. Structural similarity was further substantiated by <i>in vitro</i> binding of Cry1Aa toxin. RNA interference (RNAi) resulted in significant down-regulation of <i>AjAPN1</i> transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. <i>In vitro</i> Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of <i>A. janata</i>. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of <i>A. janata</i> was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation.</p></div

3D structures.

<p>(A) (a) AjAPN1 (model) and (b) tricorn interacting factor F3 (PDB code: 1Z1W) and (c) human endoplasmic reticulum aminopeptidase-1 (Erap1) (PDB code: 3QNF) (templates). (B) (a) <i>B. mori</i> midgut APN (model) and (b) soluble domain of human Erap1 (PDB code: 2YD0) (template). (C) Structure of APN activity and Zn⁺⁺ binding motifs of AjAPN1. The important residues are shown in ball and stick. The APN catalytic amino acid residues are shown in white backbone while Zn⁺⁺ binding amino acid residues are highlighted in pink.</p

<i>In vitro</i> Cry1Aa toxin interaction in AjAPN1 knockdown larvae.

<p>Fat body (Fb), Malpighian tubule (Mt) and salivary gland (Sg) membrane protein fractions (30 µg each) of target and control gene siRNA injected larvae were separated by 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane, then incubated with biotinylated Cry1Aa toxin (200 ng/mL) followed by further incubation with streptavidin-ALP conjugate (1∶1000 dilutions) and finally developed with NBT-BCIP substrate. Note the substantially reduced interaction of Cry1Aa toxin to the 113 kDa membrane protein of fat body and Malpighian tubule of the target gene knockdown larvae. Lower panels in each blot (β-actin expression) represent equal loading of proteins. Control (Cont): double-stranded <i>GFP</i> siRNA injected insects, Experimental (Expt): double-stranded <i>AjAPN1</i> siRNA injected insects.</p

Immunoprecipitation of Cry1Aa toxin interacting protein.

<p>Triton X-100 solubilized Malpighian tubule and salivary gland membrane protein fractions (200 µg each) prepared from early fifth instar (5E) larvae were separately incubated with purified activated Cry1Aa toxin (5 µg) followed by further incubation with Cry1Aa polyclonal antibody (2.5 µg). The Cry toxin-interacting protein complex was pull-down with Protein A agarose beads, resolved by 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane, then incubated with <i>A. janata</i> fat body APN polyclonal antibody, followed by incubation with ALP-conjugated secondary antibody and finally developed with NBT-BCIP substrate. Note the detection of a 113 kDa interacting membrane protein in both the tissues. (<b>−</b>) and (+) indicate absence and presence of Cry1Aa toxin respectively during incubation.</p