Aminopeptidase N1 is involved in Bacillus thuringiensis Cry1Ac toxicity in the beet armyworm, Spodoptera exigua

Abstract

Understanding how insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) interact with their hosts is crucial to fully explain the molecular bases of Bt specificity and insecticidal activity. Previous studies support ATP binding cassette transporters (ABCC2/3) and one cadherin-like protein are Cry1Ac functional receptors in the beet armyworm (Spodoptera exigua). In this study, a combined one-dimensional gel electrophoresis and immunoblotting approach identified aminopeptidase N (APNs) as putative Cry1Ac binding proteins in the midgut brush border membrane of S. exigua larvae. Functional analyses by gene silencing of six different S. exigua APN genes (SeAPN1, SeAPN2, SeAPN3, SeAPN4, SeAPN5 and SeAPN6) showed that only suppression of SeAPN1 resulted in decreased larval susceptibility to Cry1Ac toxin. These results support that SeAPN1 plays important functional role in Cry1Ac toxicity in S. exigua.

Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins

“…Whereas, the expression of M. sexta APN1 in S2 cells did not lead to host cell sensitivity to activated Cry1 toxins [ 32 ]. Furthermore, the down-regulated APN expression using in vivo RNAi was associated with tolerance to Cry toxins in several insects, including Spodoptera exigua APN1 [ 33 ], M. sexta APN1 [ 34 ], three Chilo suppressalis APNs [ 35 ], three Diatraea saccharalis APNs [ 36 ] and three Ae. aegypti APNs [ 37 ].…”

Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins

“…RNAi‐mediated gene silence strategy has been used to understand biological relevance of APNs to the mode of action of Bt toxins, and several lepidopteran APNs have been suggested as putative receptors for Cry1A, Cry1C or Cry2A toxins (Rajagopal et al ., ; Sivakumar et al ., ; Flores‐Escobar et al ., ; Wang et al ., ; Zhao et al ., ; Qiu et al ., , ). Because of the low interference efficiency of RNAi in lepidopteran insects (Terenius et al ., ), we need to be cautious when the RNAi technique is utilized for functional validation of candidate APNs as Bt receptors in these species.…”

Evaluation of Cry1Ac and Cry2Aa Toxin Binding to Two Important Beneficial Cotton Field Insects, Harmonia axyridis and Orius similis

“…Mass spectrometric identification of toxin binding proteins was performed using the method described previously. 26 After ligand blotting, the protein bands bound to Cry1Ac and Cry2Aa were cut and rinsed in decolorizing solution (200−400 μL, 30% acetonitrile/ 100 mM NH 4 HCO 3 ) and then freeze-dried. Later, the bands were incubated with 100 mM dithiothreitol at 56 °C for 30 min, followed by incubation with 200 mM indole-3-acetic acid for 20 min in the dark and with 100 mM NH 4 HCO 3 for 15 min at room temperature.…”

“…Brush border membrane vesicles (BBMV) from the beet armyworm, Spodoptera exigua, were used as a positive control to verify the correctness of the system adopted by us, for these BBMV were readily and specifically bind to Cry toxins , and prepared from midguts of fourth instar S. exigua larvae. , Briefly, the pooled dissected midguts were homogenized in nine volume of MET buffer A (0.3 M Mannitol, 5 mM EGTA, 17 mM Tris-HCl pH 7.5), and then one volume of 24 mM MgCl 2 was added. The homogenate was incubated on ice for 15 min, later centrifuged at 2500 g for 15 min at 4 °C.…”

“…Mass spectrometric identification of toxin binding proteins was performed using the method described previously . After ligand blotting, the protein bands bound to Cry1Ac and Cry2Aa were cut and rinsed in decolorizing solution (200–400 μL, 30% acetonitrile/100 mM NH 4 HCO 3 ) and then freeze-dried.…”