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Resistance of Trichoplusia ni to Bacillus thuringiensis toxin Cry1Ac is independent of alteration of the cadherin-like receptor for Cry toxins.

Zhang X, Tiewsiri K, Kain W, Huang L, Wang P - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York, United States of America.

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.

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The 200 kDa Cry1Ac-binding cadherin was detected in midgut BBMV proteins from both susceptible and resistant T. ni by toxin overlay binding analysis.
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pone-0035991-g003: The 200 kDa Cry1Ac-binding cadherin was detected in midgut BBMV proteins from both susceptible and resistant T. ni by toxin overlay binding analysis.

Mentions: We have previously reported that Cry1Ac bound to multiple protein bands, including the 200 kDa cadherin, from midgut BBMVs by toxin overlay binding analysis [35]. Our ligand blot binding assay in this study showed that the 200 kDa Cry1Ac-binding protein bands from the susceptible and resistant larvae were similar in staining intensity on the blot (Figure 3). Nano-LC-MS/MS analysis of the protein contents in the Cry1Ac-binding 200 kDa bands, excised from two adjacent lanes of SDS-PAGE gel loaded with equal amounts of BBMV proteins from the susceptible and the resistant larvae, respectively, identified the presence of cadherin in the protein bands and determined that the emPAI values of cadherin in the two excised gel slices from the susceptible and resistant larvae were similar (0.15 and 0.13, respectively), further indicating that the 200 kDa cadherin in the midgut BBMVs from resistant larvae was similar to that from the susceptible larvae in quantity and in binding with Cry1Ac.


Resistance of Trichoplusia ni to Bacillus thuringiensis toxin Cry1Ac is independent of alteration of the cadherin-like receptor for Cry toxins.

Zhang X, Tiewsiri K, Kain W, Huang L, Wang P - PLoS ONE (2012)

The 200 kDa Cry1Ac-binding cadherin was detected in midgut BBMV proteins from both susceptible and resistant T. ni by toxin overlay binding analysis.
© Copyright Policy
Related In: Results  -  Collection

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pone-0035991-g003: The 200 kDa Cry1Ac-binding cadherin was detected in midgut BBMV proteins from both susceptible and resistant T. ni by toxin overlay binding analysis.
Mentions: We have previously reported that Cry1Ac bound to multiple protein bands, including the 200 kDa cadherin, from midgut BBMVs by toxin overlay binding analysis [35]. Our ligand blot binding assay in this study showed that the 200 kDa Cry1Ac-binding protein bands from the susceptible and resistant larvae were similar in staining intensity on the blot (Figure 3). Nano-LC-MS/MS analysis of the protein contents in the Cry1Ac-binding 200 kDa bands, excised from two adjacent lanes of SDS-PAGE gel loaded with equal amounts of BBMV proteins from the susceptible and the resistant larvae, respectively, identified the presence of cadherin in the protein bands and determined that the emPAI values of cadherin in the two excised gel slices from the susceptible and resistant larvae were similar (0.15 and 0.13, respectively), further indicating that the 200 kDa cadherin in the midgut BBMVs from resistant larvae was similar to that from the susceptible larvae in quantity and in binding with Cry1Ac.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York, United States of America.

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.

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