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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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Schematic structures of T. ni cadherin cDNA and deduced protein sequences.(A) The cDNA (5734 bp in length) contains an open reading frame of 5202 bp from position 141 to 5342, and a poly A tail at the 3′ end. Also shown in (A) are two fragments of the genomic DNA of the cadherin gene, gDNA fragment 1 and gDNA fragment 2, amplified by PCR. gDNA PCR fragment 1 corresponds to the cDNA region from base positions 1705 to 1856 and contains an intron of 367–411 bp inserted between the cDNA base positions 1822 and 1823. gDNA PCR fragment 2 corresponds to the cDNA region from base position 4911 to 5114 and contains an intron of 291 bp inserted between cDNA base positions 4969 and 4970. (B) The deduced cadherin sequence (733 aa in length) contains a 21-aa signal peptide at the N-terminus, 11 cadherin repeats (from 1 to 11), followed by a membrane-proximal region (MPR), a transmembrane domain (TMD) of 23 amino acid residues, and a cytoplasmic domain (CPD) of 128 amino acid residues.
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pone-0035991-g001: Schematic structures of T. ni cadherin cDNA and deduced protein sequences.(A) The cDNA (5734 bp in length) contains an open reading frame of 5202 bp from position 141 to 5342, and a poly A tail at the 3′ end. Also shown in (A) are two fragments of the genomic DNA of the cadherin gene, gDNA fragment 1 and gDNA fragment 2, amplified by PCR. gDNA PCR fragment 1 corresponds to the cDNA region from base positions 1705 to 1856 and contains an intron of 367–411 bp inserted between the cDNA base positions 1822 and 1823. gDNA PCR fragment 2 corresponds to the cDNA region from base position 4911 to 5114 and contains an intron of 291 bp inserted between cDNA base positions 4969 and 4970. (B) The deduced cadherin sequence (733 aa in length) contains a 21-aa signal peptide at the N-terminus, 11 cadherin repeats (from 1 to 11), followed by a membrane-proximal region (MPR), a transmembrane domain (TMD) of 23 amino acid residues, and a cytoplasmic domain (CPD) of 128 amino acid residues.

Mentions: PCR amplification with the degenerated PCR primers designed based on known lepidopteran cadherin sequences generated a 383 bp cDNA fragment (Genbank accession no. JN849380) from the midgut cDNA library. DNA sequencing of the PCR fragment after cloning into pGEM-T vector and subsequent BLASTX search of the Genbank database indicated that the PCR fragment was a cDNA fragment of T. ni cadherin gene. Subsequent screening of the T. ni midgut cDNA library with this 383 bp fragment labeled with digoxigenin as a probe identified two cDNA clones containing a cDNA insert of 4270 bp (from nucleotide 496 to 4767, Figure S1) and 4600 bp (from nucleotide 1132 to 5732, Figure S1), respectively. A 573 bp fragment of the 5′-end of the cadherin cDNA was obtained by PCR amplification from the T. ni cDNA library with primer T3 and a cadherin specific primer to complete the 5,734 bp cadherin cDNA sequence (Genbank accession no. JF303656) (Figure S1 and Figure 1).


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)

Schematic structures of T. ni cadherin cDNA and deduced protein sequences.(A) The cDNA (5734 bp in length) contains an open reading frame of 5202 bp from position 141 to 5342, and a poly A tail at the 3′ end. Also shown in (A) are two fragments of the genomic DNA of the cadherin gene, gDNA fragment 1 and gDNA fragment 2, amplified by PCR. gDNA PCR fragment 1 corresponds to the cDNA region from base positions 1705 to 1856 and contains an intron of 367–411 bp inserted between the cDNA base positions 1822 and 1823. gDNA PCR fragment 2 corresponds to the cDNA region from base position 4911 to 5114 and contains an intron of 291 bp inserted between cDNA base positions 4969 and 4970. (B) The deduced cadherin sequence (733 aa in length) contains a 21-aa signal peptide at the N-terminus, 11 cadherin repeats (from 1 to 11), followed by a membrane-proximal region (MPR), a transmembrane domain (TMD) of 23 amino acid residues, and a cytoplasmic domain (CPD) of 128 amino acid residues.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351398&req=5

pone-0035991-g001: Schematic structures of T. ni cadherin cDNA and deduced protein sequences.(A) The cDNA (5734 bp in length) contains an open reading frame of 5202 bp from position 141 to 5342, and a poly A tail at the 3′ end. Also shown in (A) are two fragments of the genomic DNA of the cadherin gene, gDNA fragment 1 and gDNA fragment 2, amplified by PCR. gDNA PCR fragment 1 corresponds to the cDNA region from base positions 1705 to 1856 and contains an intron of 367–411 bp inserted between the cDNA base positions 1822 and 1823. gDNA PCR fragment 2 corresponds to the cDNA region from base position 4911 to 5114 and contains an intron of 291 bp inserted between cDNA base positions 4969 and 4970. (B) The deduced cadherin sequence (733 aa in length) contains a 21-aa signal peptide at the N-terminus, 11 cadherin repeats (from 1 to 11), followed by a membrane-proximal region (MPR), a transmembrane domain (TMD) of 23 amino acid residues, and a cytoplasmic domain (CPD) of 128 amino acid residues.
Mentions: PCR amplification with the degenerated PCR primers designed based on known lepidopteran cadherin sequences generated a 383 bp cDNA fragment (Genbank accession no. JN849380) from the midgut cDNA library. DNA sequencing of the PCR fragment after cloning into pGEM-T vector and subsequent BLASTX search of the Genbank database indicated that the PCR fragment was a cDNA fragment of T. ni cadherin gene. Subsequent screening of the T. ni midgut cDNA library with this 383 bp fragment labeled with digoxigenin as a probe identified two cDNA clones containing a cDNA insert of 4270 bp (from nucleotide 496 to 4767, Figure S1) and 4600 bp (from nucleotide 1132 to 5732, Figure S1), respectively. A 573 bp fragment of the 5′-end of the cadherin cDNA was obtained by PCR amplification from the T. ni cDNA library with primer T3 and a cadherin specific primer to complete the 5,734 bp cadherin cDNA sequence (Genbank accession no. JF303656) (Figure S1 and Figure 1).

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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