ABSTRACT
Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.
Subject(s)
Animals , Bacillus thuringiensis , Bacterial Proteins , Metabolism , Bombyx , CD13 Antigens , Metabolism , Cadherins , Metabolism , Endotoxins , Metabolism , Hemolysin Proteins , Metabolism , LarvaABSTRACT
Aminopeptidase N (APN) belonging to zinc-dependent metalloproteinase, not only catalyzes protein proteolytic process, but also is involved in the pathogenic process as the receptor of pathogenic toxin. In Bombyx mori, APN gene family consists of 16 members, of which BmAPN4 binds trypsin-activated parasporal crystal (PC) toxin isolated from Bacillus bombysepticus (Bb). In order to verify whether or not other APNs interact with PC toxin during the pathogenesis of Bb, we cloned BmAPN5, a member of aminopeptidase family, from the silkworm midgut. The full length of BmAPN5 is 3313 bp, encoding 953 amino acids, containing a zinc peptidase_M1 and ERAP1_C domains. A recombinant GST-BmAPN5 was purified by a prokaryotic expression system. Far-Western blotting, co-immunoprecipitation and ELISA. Binding saturation assays demonstrated that PC after activated by trypsin could be bound by BmAPN5. Additionally, cytotoxic activity of trypsin-activated PC in Sf9 cells transfected with BmAPN5 showed that cells exhibited dramatic cytological changes, including swelling and lysis, revealing BmAPN5 serves as a functional receptor that participates in Bb and PC pathogenicity. These provide some clues for further exploring the pathogenesis relationships of Bb and host.
ABSTRACT
Fhx/P25 in silkworm, Bombyx mori, one of the main components of silk fibroin, is presumed in previous reports to be expressed exclusively in the posterior silk gland (PSG) of the animal with strict territorial and developmental specificities. On the basis of a large-scale analysis ofthe silkworm EST data, it was found that Fhx/P25 gene is transcribed not only in the posterior silk gland, but in the ovary and in other tissues of the larvae at day 3 of the fifth-instar as well and that this gene has distinct transcription start sites (TSSs) in the posterior silk gland and the ovary. The TSS in the ovary is located about 115 bp upstream sequence of that in the posterior silk gland. Subsequent RT-PCR, FQ-PCR and sequencing have verified the validity of this presumption. In addition, alternative splicing is predicted in pre-mRNA of Fhx/P25 gene and confirmed by RT-PCR. In conclusion, Fhx/P25 gene is not a gene with strictly tissue-specific transcription.Complicated regulation mechanisms may exist for its transcription and expression and it may have other functions to perform.