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United States Department of Agriculture

Agricultural Research Service

Research Project: INTEGRATED PEST MANAGEMENT FOR MID-SOUTH AREA ROW CROPS Title: Molecular characterization and RNA interference of three midgut aminopeptidase N isozymes from bacillus thuringiensis-susceptible and -resistant strains of sugarcane borer diatraea saccharalis

Authors
item Yang, Yunlong -
item Zhu, Yu Cheng
item Abel, Craig
item Ottea, James -
item Husseneder, Claudia -
item Leonard, Roger -
item Huang, Fangneng -

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 27, 2010
Publication Date: July 14, 2010
Citation: Yang, Y., Zhu, Y., Abel, C.A., Ottea, J., Husseneder, C., Leonard, R.B., Huang, F. 2010. Molecular characterization and RNA interference of three Midgut Aminopeptidase N Isozymes from bacillus thuringiensis-susceptible and -resistant strains of sugarcane borer, diatraea saccharalis. Insect Biochemistry and Molecular Biology. 40:592-603.

Interpretive Summary: Since first being commercialized in 1996, transgenic corn plants expressing Cry1Ab toxin have been the most widely grown Bt corn for controlling corn stalk boring pests in the United States and several other countries. The sugarcane borer is a major corn pest and a primary target of Bt corn in the mid-south region of the United States. Recently, a strain resistant to Cry1Ab protein in Bt corn was identified from a field population of the sugarcane borer. This Bt-resistant strain completed its entire larval development (from neonate to pupa stages) on commercial Cry1Ab corn hybrids. The objective of this study was to determine if Cry1Ab resistance in the sugarcane borer was associated with changes in gene structure/expression of aminopeptidase N (APN). The APNs are proteins located at the midgut epithelium of some lepidopterous species that may be implicated as receptors for insecticidal Bt toxin. cDNAs (gene transcript) of three APN isoforms, DsAPN1, DsAPN2, and DsAPN3, from the susceptible and resistant strains of the sugarcane borer were identified and sequenced in this study. Results showed that cDNA sequences of the three APN genes were identical between the susceptible and resistant strains. However, total APN proteolytic activity and gene expression of the three APNs from the resistant larvae were significantly lower than those of the susceptible strain. RNA interference (RNAi) was employed using an oral droplet feeding technique for the three APNs of the susceptible strain. Down-regulating expressions of the three APN genes by RNAi were correlated with the reductions in the specific APN activity. In addition, silencing of all three APNs in the sugarcane borer by RNAi resulted in a decrease in Cry1Ab susceptibility. Our results showed that reduction in expression of the three APNs is functionally associated with the Cry1Ab resistance in the sugarcane borer.

Technical Abstract: Aminopeptidase N (APN) proteins located at the midgut epithelium of some lepidopterous species have been implicated as receptors for insecticidal proteins from Bacillus thuringiensis. cDNAs of three APN isoforms, DsAPN1, DsAPN2, and DsAPN3, from Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-RR) strains of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), were identified and sequenced using reverse transcriptase polymerase chain reaction (RT-PCR) and 5’ rapid amplification of cDNA end (5’ RACE). The characteristic APN sequence features were derived from deduced amino acid sequences of the cloned cDNAs. cDNA sequences of the three APN genes were identical between the Cry1Ab-SS and -RR strains. However, total APN proteolytic activity and gene expression of the three APNs from Cry1Ab-RR larvae were significantly lower than those of the Cry1Ab-SS strain. RNA interference (RNAi) was employed using an oral droplet feeding technique for the three APNs of the Cry1Ab-SS strain. Down-regulating expressions of the three APN genes by RNAi were correlated with the reductions in the specific APN activity. In addition, silencing of all three APNs in D. saccharalis in vivo by RNAi resulted in a decrease in Cry1Ab susceptibility. Our results showed that reduction in expression of the three APNs is functionally associated with the Cry1Ab resistance in D. saccharalis.

Last Modified: 12/21/2014
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