title: RNA interference-mediated knockdown of three putative aminopeptidases N affects susceptibility of Spodoptera exigua larvae to Bacillus thuringiensis Cry1Ca.
abstract: Aminopeptidase N (APN) isoforms in insects have been documented to be involved in the mode of action of insecticidal crystal proteins (Cry) from Bacillus thuringiensis. Here we cloned two novel Seapns from the larval midgut of Spodoptera exigua, a major pest of many crops of economic importance in China. According to a phylogenetic analysis, these two novel SeAPNs, along with the four SeAPN isoforms already described, belong to six different clades. All the six SeAPNs share similar structural features. From N- to C-terminus a signal peptide, a gluzincin aminopeptidase motif, a zinc binding/gluzincin motif, and a glycosylphosphatidylinositol-anchor sequence are located. The six Seapn genes were highly expressed at the larval stage, especially in the larval gut. Ingestion during four consecutive days of double-stranded RNAs (dsRNAs) targeting Seapn1, Seapn2, Seapn3, Seapn4, Seapn5 and Seapn6 significantly reduced corresponding mRNA levels by 55.6%, 45.5%, 43.2%, 56.8%, 45.4%, and 46.0% respectively, compared with those recorded in control larvae fed on non-specific dsRNA (dsegfp). When the larvae that previously ingested phosphate buffered saline (PBS)-, dsegfp-, or six dsSeapns-overlaid diets were then exposed to a diet containing Cry1Ca, the larval mortalities were 71.2%, 69.3%, 52.0%, 77.2%, 43.3%, 62.0%, 65.4% and 53.8% respectively recorded after 6days. ANOVA analysis revealed that the larvae previously fed on dsSeapn1-, dsSeapn3-, and dsSeapn6-overlaid diets had significantly lower mortalities than those previously ingested PBS-, dsegfp-, dsSeapn2-, dsSeapn4- and dsSeapn5-overlaid diets. Thus, these results suggest that SeAPN1, SeAPN3 and SeAPN6 may be candidate receptors for Cry1Ca in S. exigua.
authors.affiliation: Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China.
authors.forename: Xiang-Liang
authors.initials: XL
authors.lastname: Ren
authors.affiliation: Institute of Cotton Research of CAAS, State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Institute of Cotton Research of CAAS, State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China.
authors.forename: Jin-Jie
authors.initials: JJ
authors.lastname: Cui
authors.affiliation: Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China. Electronic address: ligq@njau.edu.cn.
authors.forename: Guo-Qing
authors.initials: GQ
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism; D001426:Bacterial Proteins / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology*; D018826:CD13 Antigens / Q000096:biosynthesis* / Q000235:genetics*; D015870:Gene Expression; D007814:Larva / Q000187:drug effects* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010572:Pest Control, Biological; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000187:drug effects / Q000378:metabolism; D017422:Sequence Analysis, DNA; D018411:Spodoptera / Q000187:drug effects* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D012330:RNA, Double-Stranded; D018826:CD13 Antigens
keywords: Aminopeptidase N; Bacillus thuringiensis; Candidate receptor; Crystal toxin; RNA interference; S. exigua