Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes.
BACKGROUND
Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs) from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of four APN cDNAs from Spodoptera exigua.


RESULTS
Suppression Subtractive Hybridization (SSH) was used to construct cDNA libraries of genes that are up-and down-regulated in the midgut of last instar larvae of beet armyworm, S. exigua exposed to B. thuringiensis Cry1Ca toxin. Among the clones from the SSH libraries, cDNA fragments coding for two different APNs were obtained (APN2 and APN4). A similar procedure was employed to compare mRNA differences between susceptible and Cry1Ca resistant S. exigua. Among the clones from this last comparison, cDNA fragments belonging to a third APN (APN1) were detected. Using sequences obtained from the three APN cDNA fragments and degenerate primers for a fourth APN (APN3), the full length sequences of four S. exigua APN cDNAs were obtained. Northern blot analysis of expression of the four APNs showed complete absence of APN1 expression in the resistant insects, while the other three APNs showed similar expression levels in the resistant and susceptible insects.


CONCLUSIONS
We have cloned and characterized four different midgut APN cDNAs from S. exigua. Expression analysis revealed the lack of expression of one of these APNs in the larvae of a Cry1Ca-resistant colony. Combined with previous evidence that shows the importance of APN in the mode of action of B. thuringiensis toxins, these results suggest that the lack of APN1 expression plays a role in the resistance to Cry1Ca in this S. exigua colony.
Business Unit Bioscience, Plant Research International B.V., Wageningen University and Research Center, The Netherlands. salvador.herrero@uv.es
Salvador
S
Herrero
Tsanko
T
Gechev
Petra L
PL
Bakker
William J
WJ
Moar
Ruud A
RA
de Maagd
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D015152:Blotting, Northern; D018826:CD13 Antigens / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004731:Endotoxins / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D015723:Gene Library; D006460:Hemolysin Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010572:Pest Control, Biological; D010802:Phylogeny; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012984:Software; D018411:Spodoptera / Q000378:metabolism*; D015854:Up-Regulation
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D018076:DNA, Complementary; D004731:Endotoxins; D006460:Hemolysin Proteins; D012333:RNA, Messenger; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens