Abstract
Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin alteration.
Highlights
The soil bacterium Bacillus thuringiensis (Bt) is the most successfully used microbial insect control agent in agriculture and public health [1,2]
Results from this study indicate that Bt-resistance evolved in the greenhouse populations of T. ni is independent of alteration of the midgut cadherin, which is different from the cadherin mutation-associated genetic basis of ‘‘Mode 1’’ type resistance
Results cDNA Sequence of the T. ni Cadherin Gene PCR amplification with the degenerated PCR primers designed based on known lepidopteran cadherin sequences generated a 383 bp cDNA fragment
Summary
The soil bacterium Bacillus thuringiensis (Bt) is the most successfully used microbial insect control agent in agriculture and public health [1,2]. Development of resistance to Bt toxins in insect populations threatens the sustainable application of both sprayable Bt-based biopesticides and Bt-crops for insect control. The potential for development of insect resistance to Bt toxins has been widely demonstrated by successful establishment of various Bt-resistant insect populations through selection with Bt toxins under laboratory conditions [4,5]. Cases of insect resistance to Bt biopesticides and Bt-crops have been reported in six lepidopteran species [6,7,8,9,10]. To achieve continuing success in application of Bt-based technologies for insect pest control, it is crucially important to understand the molecular genetics of Bt resistance evolved in insect populations in agricultural systems, which is currently unclear for any case of field- or greenhouseevolved Bt-resistance
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