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Proc Natl Acad Sci U S A. Aug 23, 2011; 108(34): 14037–14042.
Published online Aug 15, 2011. doi:  10.1073/pnas.1102555108
PMCID: PMC3161562
Agricultural Sciences

Differential alteration of two aminopeptidases N associated with resistance to Bacillus thuringiensis toxin Cry1Ac in cabbage looper

Abstract

The soil bacterium Bacillus thuringiensis (Bt) is the most successfully used biopesticide in agriculture, and its insecticidal protein genes are the primary transgenes used for insect control in transgenic crops. However, evolution of insect resistance to Bt toxins threatens the long-term future of Bt applications. To date, cases of resistance to Bt toxins have been reported in agricultural situations in six insect species, but the molecular basis for these cases of resistance remains unclear. Here we report that the resistance to the Bt toxin Cry1Ac in the cabbage looper, Trichoplusia ni, evolved in greenhouses, is associated with differential alteration of two midgut aminopeptidases N, APN1 and APN6, conferred by a trans-regulatory mechanism. Biochemical, proteomic, and molecular analyses showed that in the Cry1Ac-resistant T. ni, APN1 was significantly down-regulated, whereas APN6 was significantly up-regulated. The Cry1Ac resistance was correlated with down-regulation of APN1 but not with the up-regulation of APN6. The concurrent up-regulation of APN6 and down-regulation of APN1 might play a role in compensating for the loss of APN1 to minimize the fitness costs of the resistance. Along with identifying reduced expression of APN1 as the molecular basis of Bt resistance selected in an agricultural setting, our findings demonstrate the importance of APN1 to the mode of action of Bt toxin Cry1Ac.

The soil bacterium Bacillus thuringiensis (Bt) has long been used as a biopesticide for insect control in agriculture and public health (1, 2). Bt genes coding for insecticidal toxins are the primary transgenes in current transgenic crops (Bt crops) conferring insect resistance (3). With the increasing scale and prolonged planting of Bt crops, evolution of insect resistance to Bt toxins in agricultural systems has become the foremost threat to the long-term future of Bt biopesticides and Bt crops. To date, cases of insect resistance to Bt toxins in open fields or greenhouses have been reported in six species (2).

Cry toxins are the major insecticidal proteins in Bt. Once ingested by insects, the Cry protoxins are activated by the insect digestive proteases. The active toxins penetrate through the insect midgut peritrophic membrane and reach the midgut brush border membrane, where they interact with specific binding sites, leading to cell lysis after a multistep cascade that is incompletely understood. The Bt pathogenesis pathway is complex, and thus mechanisms of Bt resistance can be diverse. To date, numerous insect strains resistant to Bt toxins have been established by selection with Bt toxins under laboratory conditions (4), and various resistance mechanisms have been reported in laboratory-selected Bt-resistant insects, including alterations of midgut digestive proteases, decreased peritrophic membrane permeability, heightened immune response, enhanced esterase production, and reduced Cry toxin binding (1, 2, 5). More recently, mutation of an ABC transporter in Heliothis virescens was found to correlate with resistance to Cry1A toxins (6). However, it has become clear that resistance mechanisms in laboratory-selected Bt-resistant insects do not necessarily represent Bt resistance mechanisms evolved in field populations (7). Molecular mechanisms of Bt resistance evolved in agriculture have not yet been reported.

“Mode 1” type resistance is the most common type of resistance to Bt observed in insects. It is characterized by a high level of resistance (>500-fold) to at least one Cry1A toxin but not to Cry1C toxins, recessive inheritance, and reduced binding of one or more Cry1A toxins to the midgut brush border membrane (8). Mode 1 type resistance has been associated with mutations of the midgut cadherin gene in three Lepidoptera: H. virescens, Pectinophora gossypiella, and Helicoverpa armigera (911). The midgut cadherin has been suggested to serve as the first Cry toxin receptor in the sequential interactions of Cry toxins with midgut brush border membrane proteins (12, 13). Consequently, mutations in the cadherin gene may result in Bt resistance in insects (911). The field- and greenhouse-evolved resistance to Cry1Ac in Plutella xylostella and T. ni is of the typical mode 1 type. However, the cadherin gene in P. xylostella or T. ni is not involved in the resistance mechanism (14, 15), indicating that the mode 1 type resistance selected in agricultural systems involves a different but yet to be known molecular genetic mechanism. The Cry1Ac resistance that has evolved in T. ni populations in commercial greenhouses is monogenic and recessive in inheritance and is conferred by loss of toxin-binding sites in the larval midgut brush border membrane (16, 17). In this study, the greenhouse-derived Cry1Ac-resistant T. ni (16) was used as a unique system to identify the alteration of Cry1Ac-binding proteins and its association with the resistance to Cry1Ac. This study reports the identification of the molecular basis of insect resistance to Bt toxins evolved in an agricultural system.

Results

Alteration of Midgut Brush Border Membrane Vesicle Proteins in Cry1Ac-Resistant Larvae Identified by SDS/PAGE Separation.

Proteins from the midgut brush border membrane vesicle (BBMV) proteins of the susceptible Cornell strain and the resistant GLEN-Cry1Ac-BCS strain exhibited highly similar protein profiles on SDS/PAGE analysis, except for the absence of a protein band at 110 kDa in BBMVs from resistant larvae (Fig. 1). In this band, APN1 was the primary protein with 8 and 46 minor proteins, respectively, determined by nano liquid chromatography–tandem mass spectroscopy (nano LC-MS/MS) analyses with two different mass spectrometry systems, the Synapt HDMS (Waters) and the LTQ Orbitrap Velos (Thermo-Fisher Scientific). The relative abundance of APN1 in this 110-kDa band was estimated as 77 and 42 mol %, respectively, with the data obtained from the two mass spectrometry systems using exponentially modified protein abundance index (emPAI) values of the identified proteins [protein content (mol %) = emPAI/∑(emPAI) × 100] (18). Further identification of proteins from 17 protein bands ranging from 33 to >250 kDa from the susceptible strain and 8 matching bands ranging from 88 to >250 kDa from the resistant strain showed that APN6 was widely present in the protein bands analyzed from the resistant strain, in abundances ranging from 0.03 to 2.7 mol %. However, among the 17 protein bands analyzed from the susceptible strain, only one band minimally visible on the gel at 150 kDa was found to contain APN6 at low abundance (0.03 mol %) (Fig. 1B).

Fig. 1.
SDS/PAGE analysis of midgut BBMV proteins from Cry1Ac-resistant and -susceptible larvae. (A) BBMV proteins from the resistant larvae lacked a 110-kDa protein band. APN1 was identified to be the primary protein in the band, accounting for 77 and 42 mol ...

Alteration of Midgut BBMV Proteins in Cry1Ac-Resistant Larvae Identified by Liquid-Based Quantitative Proteomic Analysis.

Comparative proteomic analysis of midgut BBMV proteins from the susceptible and resistant larvae by isobaric tagging for relative and absolute quantification (iTRAQ) identified 1,464 and 1,440 proteins at a 95% confidence interval in two independently prepared sample sets, including 908 proteins identified in both sample sets. Of these 908 proteins, those containing at least one unique peptide (496 proteins) were used for quantitative analysis to ensure quantitative data reliability. Based on the variations observed between the two sample sets [Δlog2 ratio = │log2 ratio(sample set 1) − log2 ratio(sample set 2)│], the iTRAQ quantitative ratio [│log2 (115 + 117)/(114 + 116)│] cutoff point was determined to be 1.5 (i.e., 95% of the proteins identified fell within a deviation of 1.5). With a significance cutoff threshold of 1.5, only two proteins, APN1 and APN6, were identified to be significantly different in quantity between the susceptible and the resistant strains in both sample sets. The log2ratio of APN1 in the BBMV proteins between the resistant and susceptible strains was found to be −3.1 and −3.3 in the two sample sets. Therefore, the ratio of APN1 between the resistant and susceptible strains was 0.11 (quantitative ratio = 2[log2ratio(sample set 1) + log2ratio(sample set 2)]/2). In contrast, the quantity of APN6 was higher in the BBMV proteins of the resistant strain. The log2ratio of APN6 between the resistant and susceptible strains was 2.6 in both sample sets; therefore, the ratio of APN6 between the resistant and susceptible strains was 6.0.

Midgut BBMV Proteins from Cry1Ac-Resistant Larvae Lack the 110-kDa Cry1Ac- Binding Protein APN1.

A Cry1Ac toxin overlay binding analysis of the midgut BBMV proteins showed that Cry1Ac could bind to multiple proteins on the membrane blot, and that the patterns for the samples from the resistant and susceptible larvae were similar, except for the lack of a positive band at 110 kDa from the resistant larvae (Fig. 2A). Identification of the proteins in this 110-kDa band by nano LC-MS/MS confirmed that APN1 was the primary protein (77 mol %). The positive band at 200 kDa was not visible on the SDS/PAGE gel stained with Coomassie blue, but analysis of the gel slice corresponding to the position of the positive band on the membrane blot showed that the band contained cadherin. The primary protein in the >200-kDa positive band was identified as polycalin, which accounted for 26 mol % of the proteins in the band by emPAI-based estimation. Western blot analysis of the midgut BBMV proteins confirmed that the 110-kDa Cry1Ac-binding protein in the midgut BBMVs was APN1, and that this 110-kDa APN1 was lacking in the resistant larvae (Fig. 2B).

Fig. 2.
Cry1Ac toxin overlay binding analysis and identification of toxin-binding proteins from midgut BBMV proteins. (A) Three Cry1Ac-binding proteins from the midgut BBMV proteins were identified as polycalin, cadherin, and APN1 by nano LC-MS/MS. The 110-kDa ...

Transcript Levels of APN1 and APN6 Genes Are Differentially Altered in Cry1Ac-Resistant Larvae.

Quantitative RT-PCR (qRT-PCR) analysis revealed a significantly lower transcript level of apn1 in the resistant strain, with a ratio of 0.026 to the transcript level of apn1 in the susceptible strain. In contrast, the transcript level of apn6 in the resistant strain was significantly up-regulated, with a ratio of 39 to that in the susceptible strain. The transcript levels of apn2, apn3, apn4, and apn5 did not differ significantly between the susceptible and resistant larvae (Fig. 3).

Fig. 3.
Relative transcript levels of APN genes in the Cry1Ac-resistant and -susceptible larvae by qRT-PCR analysis. Error bars indicate SEMs from the analysis of five individuals. Asterisks indicate that transcript levels in the resistant larvae were significantly ...

APN1 and APN6 Genes Show No Genetic Linkage with Cry1Ac Resistance.

For linkage analysis of apn1 and apn6 with Cry1Ac resistance, a single-pair cross between a male from the GLEN-Cry1Ac-BCS strain (apn1Gapn1G apn6Gapn6G) and a female from the Benzon strain (apn1Bapn1B apn6Bapn6B) was done to produce F1 progeny (apn1Gapn1B apn6Gapn6B), after which a female F1 progeny was backcrossed with a male from the GLEN-Cry1Ac-BCS strain to produce backcross progenies. Genotyping of 12 Cry1Ac-selected larvae and 12 Cry1Ac-nonselected larvae from the backcross family showed that in both the Cry1Ac-selected and -nonselected groups, six individuals had the genotype apn1Gapn1B apn6Gapn6B, and the other six individuals had the genotype apn1Gapn1G apn6Gapn6G. The ratios of apn1Gapn1G to apn1Gapn1B and of apn6Gapn6G to apn6Gapn6B were 1:1, as would be expected with perfect random assortment (P > 0.10 by the χ2 test), in both the Cry1Ac-selected and -nonselected groups, demonstrating that apn1 and apn6 did not cosegregate with the resistance trait. This indicates that there is no genetic linkage between apn1 and apn6 with Cry1Ac resistance. Neither genotype apn1Gapn1G apn6Gapn6B nor apn1Gapn1B apn6Gapn6G was present in the 24 individuals analyzed, indicating that apn1 and apn6 are in the same linkage group, as is known in other Lepidoptera (19).

Association Between Resistance and Transcription of APN Genes.

Two reciprocal F2 backcross families were generated between the GLEN-Cry1Ac-BCS strain and its near-isogenic Cornell strain and treated with or without Cry1Ac selection. qRT-PCR analysis of apn1 transcript levels in larval midgut showed that in both backcross families, the non–Cry1Ac-selected individuals clearly exhibited two distinct groups in apn1 transcript level (Fig. 4). One group demonstrated a significantly reduced apn1 transcript level to a ratio of no more than 0.1 to the susceptible strain and no detectable 110-kDa APN1 by Western blot analysis. The other group had an apn1 transcript level ratio of >0.4 to that of the susceptible strain, which was similar to that in F1 individuals, and APN1 was detected by Western blot analysis. The ratio between the numbers of individuals of the two groups was 8:9 in both families, which was statistically similar to the predicted random assortment ratio 1:1 (P > 0.10 by the χ2 test). In contrast, all of the larvae in the Cry1Ac-selected groups from both backcross families (22 larvae in all) had a significantly reduced apn1 transcript level with a ratio <0.1 to the susceptible larvae (Fig. 4), demonstrating the linkage (cosegregation) between the significantly reduced apn1 transcript level (apn1 transcript level <0.1) and Cry1Ac resistance (P < 0.001, χ2 test). APN6 gene transcript levels in both Cry1Ac-selected and -nonselected larvae were similarly up-regulated, with a ratio to that of the susceptible strain ranging from 15 to >75, with no association with resistance (Fig. 4). Although the unselected backcross individuals in both backcross families exhibited two distinct groups with differing apn1 transcript levels and differing presence or absence of APN1 (Fig. 4), these two groups of larvae had similar apn6 transcript levels (P > 0.10, t test of the means of apn6 transcript levels in the two groups). Therefore, there was no correlation between the levels of apn1 and apn6 transcripts in the larvae.

Fig. 4.
Transcript levels of APN1 and APN6 genes in F1, Cry1Ac-selected and -nonselected larvae from backcross family a (A) and family b (B) analyzed by qRT-PCR. The transcript levels are relative to the transcript levels in the susceptible Cornell strain. The ...

Discussion

In this study, comparative biochemical and proteomic analyses have shown that the Cry1Ac-resistant T. ni strain differs from its near-isogenic susceptible strain by two proteins in the midgut brush border membrane, APN1 and APN6. In the Cry1Ac-resistant strain, APN1 expression was significantly reduced to a ratio of 0.11 at the protein level and a ratio of 0.026 at the transcript level to the susceptible strain. In contrast, APN6 was significantly increased, to a ratio of 6.0 at the protein level and a ratio of 39 at the transcript level. Furthermore, the 110-kDa APN1 protein was lacking in the resistant larvae (Figs. 1 and 2). The differential changes of APN1 and APN6 proteins in the resistant strain were not conferred by mutations of the APN1 and APN6 genes, but instead were regulated at the transcription level by an as-yet unidentified trans-regulatory mechanism (Fig. 3). The down-regulation of APN1 expression was linked to the resistance to Cry1Ac in T. ni (Fig. 4).

Recent studies have indicated that Cry1A toxins interact with at least two types of receptors on the midgut brush border membrane (12, 13, 20, 21). Activated Cry1A toxins bind to the first receptor (the midgut cadherin) with high affinity, and the interaction with cadherin facilitates oligomerization of the toxins via a proteolytic process. The Cry1A oligomers have a high binding affinity to the secondary receptor (APN or alkaline phosphatase) and thus bind to the secondary receptor, eventually leading to insertion of the oligomers into the midgut cell membrane, with resulting cell lysis. It also has been proposed that binding of Cry1A toxins to the cadherin may activate a cellular signaling pathway leading to cell death without the involvement of APN (21). Nevertheless, in the sequential toxin-binding events, cadherin has been suggested to serve as the first receptor for Cry1A toxins. Loss-of-function (i.e., binding affinity to Cry1A) mutations of the cadherin gene are expected to confer mode 1 type resistance as observed in H. virescens, P. gossypiella, and H. armigera (911); however, the mode 1 type resistance in T. ni is associated with a loss of APN1. In previous studies, we found that specific binding of Cry1Ab or Cry1Ac to the midgut BBMVs from the Cry1Ac-resistant T. ni larvae was undetectable, and that cadherin was not involved in the resistance (15, 16). Therefore, results from our studies on Cry1Ac resistance in T. ni indicate that cadherin alone without APN1 is not sufficient to serve as an efficient binding site for Cry1Ab or Cry1Ac toxins in the midgut of T. ni, and that the toxicity of Cry1Ab or Cry1Ac in T. ni requires crucial involvement of APN1.

APN1 has been well documented as a Cry1A-binding protein associated with toxicity of Cry1A toxins in lepidopterans (20). The Cry1Ac overlay binding analysis in this study confirmed Cry1Ac binding to APN1 of T. ni (Fig. 2). More recently, APN6 has been identified from lepidopteran larvae, and affinity purification of H. armigera midgut proteins using Cry1Ac as a ligand indicated that APN6 was not among the Cry1Ac-binding proteins (22). Similarly, results of the present study indicate that APN6 in the midgut BBMVs from the Cry1Ac-resistant T. ni is significantly more abundant than that from the Cry1Ac-susceptible strain (Fig. 1), but no specific binding of Cry1Ac or Cry1Ab to the midgut BBMVs from resistant larvae could be detected (16), nor additional midgut BBMV protein bands with Cry1Ac binding affinity were detected from resistant larvae by the Cry1Ac overlay binding assay (Fig. 2). Therefore, APN6 does not appear to be a Cry1A-binding protein. In a recent report, Pacheco et al. (23) proposed a “ping-pong” binding mechanism based on observations on the binding of Cry1Ab mutants to Manduca sexta cadherin and APN. In this ping-pong binding model, the Cry toxin first binds to the abundant but low-affinity APN, facilitating concentration of the toxin at the midgut brush border membrane for the consequent binding to cadherin. The Cry toxin binds to the APN with a high affinity after oligomerization on interaction with cadherin (23). Our results indicate that binding of Cry1Ab or Cry1Ac to the midgut brush border membrane in T. ni requires APN1, because no specific binding sites for Cry1Ab or Cry1Ac could be detected in the midgut BBMVs from the Cry1Ac-resistant T. ni (16).

It has been well documented that Cry1Ab or Cry1Ac can bind to isolated cadherin protein without APN (12, 24, 25). Cry1Ac also can bind to the cadherin from both the Cry1Ac-susceptible and the Cry1Ac-resistant T. ni larvae, as demonstrated by the toxin overlay binding assay (Fig. 2), in disagreement with the previous observation that Cry1Ac does not bind to the BBMVs from the resistant T. ni (16). Apparently, the binding of toxin to cadherin in the midgut brush border membrane differs from that of the separated proteins in vitro.

Since the first report of field-evolved insect resistance to Bt in 1990 (26), populations of two insect species, P. xylostella, and T. ni, have developed resistance to Bt sprays in open-field or commercial greenhouses, and four other species, Busseola fusca, Spodoptera frugiperda, Helicoverpa zea, and P. gossypiella, have developed resistance to Bt crops (2, 3). However, the molecular mechanisms of the field- and greenhouse-evolved resistance have not yet been identified. Here we report the identification of a molecular mechanism of Bt resistance evolved in agricultural systems. With alteration of APNs as the molecular basis for Cry1Ac resistance, the greenhouse-evolved T. ni not only is highly resistant to Cry1Ac on an artificial diet, but also is highly resistant to Cry1Ac plants (16). Interestingly, different alleles and differing transcript levels of APN genes have been reported in laboratory-selected Cry1A toxin-resistant strains of several lepidopterans (27, 28). However, genetic linkages between the different APN alleles or differential transcript levels and the resistance in these insects have not been established to confirm the correlation with resistance.

The heterozygous individuals and the homozygous Cry1Ac-resistant individuals clearly showed significantly different levels of apn1 expression (unselected individuals in Fig. 4). However, the expression level of apn6 in these same individuals did not demonstrate a correlated pattern (Fig. 4). Therefore, the up-regulated apn6 expression is unlikely to be a response to the reduction of apn1 expression, but may be coregulated with apn1 expression. The factor or factors regulating the differential transcription of apn1 and apn6 remain to be identified. The possibility that two different but genetically linked pathways regulate the expression of apn1 and apn6 cannot be excluded. Although up-regulation of apn6 transcription is not directly linked to Cry1Ac resistance, the up-regulation of apn6 expression could play a role in minimizing possible fitness costs associated with the resistance by compensating for the loss of APN1 with APN6. Indeed, midgut aminopeptidase activities were similar in the resistant and susceptible T. ni (16).

The genetic basis for the alteration of Cry1A-binding sites in mode 1 type resistance is reportedly associated with target site (cadherin) gene mutations (911). Our present results show that Bt toxin-binding site alteration can be conferred by a trans-regulatory mechanism as well. Therefore, the molecular bases for mode 1 type high-level Bt resistance are not limited to binding site mutations, and the genetic bases for Bt resistance are more diverse than previously thought.

Materials and Methods

Insects.

A highly inbred laboratory strain of T. ni (Cornell strain) was used as the susceptible strain (17). A Cry1Ac-resistant strain of T. ni near-isogenic to the susceptible Cornell strain, GLEN-Cry1Ac-BCS, was derived from a Bt-resistant population collected from commercial greenhouses (16). The GLEN-Cry1Ac-BCS strain had been backcrossed with the Cornell strain eight times at the time that study was carried out. In addition, a susceptible T. ni strain from Benzon Research (Benzon strain) was used for genetic linkage analysis of APN genes with resistance to Cry1Ac in T. ni.

Preparation of Bt Toxin Cry1Ac.

Cry1Ac protoxin was prepared from the Bt kurstaki strain HD-73, as described by Kain et al. (17). The Cry1Ac protoxin was activated by incubation with N-p-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin at a ratio of 1: 20 (wt/wt) in 50 mM Na2CO3 (pH 10.0) at 37 °C for 1–16 h, and completion of digestion was examined by SDS/PAGE analysis. Activated Cry1Ac toxin was purified by anion-exchange chromatography on a UNO Q column (Bio-Rad) with a linear gradient of 0–1 M NaCl in 20 mM Na2CO3 (pH 10.0).

Midgut BBMV Preparation.

Mid-fifth instar larvae were dissected in cold dissection buffer [17 mM Tris-HCl (pH 7.5), 5 mM EGTA, 300 mM mannitol, 1 mM PMSF] to isolate the midgut epithelium. Midgut BBMV proteins were prepared as described by Wolfersberger et al. (29). The protein concentrations of the BBMV protein preparations were determined using the Bio-Rad Protein Assay Kit II. The activities of brush border membrane marker enzymes alkaline phosphatase and aminopeptidase in the BBMV protein preparations and their initial midgut tissue homogenates were determined (30) to confirm the enrichment of brush border membranes in the BBMV protein preparations. The enrichment of the two enzyme activities was typically 5- to 6-fold and 7- to 10-fold, respectively.

iTRAQ Labeling and Quantitative Proteomic Analysis of Midgut BBMV Proteins.

BBMV proteins were solubilized in 0.5 M Hepes (pH 7.4) with 5 mM EGTA, 0.3 M mannitol, and 1% SDS and then reduced with 5 mM Tris-(2-carboxyethyl)-phosphine at 37 °C for 1 h. Subsequently, the thiol groups were blocked with 8 mM methyl methanethiosulfonate at room temperature for 10 min. The protein samples were then digested with sequencing-grade modified trypsin at 37 °C for 16 h, and the resulting tryptic peptides were labeled using iTRAQ reagent (Applied Biosystems). The sample from the Cornell strain was labeled separately with reporter ion reagents 114 and 116, and that from the GLEN-Cry1Ac-BCS strain was labeled with reporter ion reagents 115 and 117 as technical repeats. The labeled samples were combined and fractionated by OFFGEL isoeletric focusing electrophoresis using the Agilent 3100 OFFGEL Fractionator (Agilent) with Immobiline DryStrip (pH 3–10; 24 cm) (GE Healthcare). The fractions collected were pooled into 10 final fractions and analyzed by nano LC-MS/MS analysis after desalting by solid-phase extraction using the Sep-Pak C18 cartridge (Waters).

Nano LC-MS/MS analysis was performed using the LTQ Orbitrap Velos mass spectrometer equipped with nano ion source with high-energy collisional dissociation at Cornell University's Proteomics and Mass Spectrometry Core Facility. Samples were injected onto a PepMap C18 trap column (5 μm; 300 μm × 5 mm) (Dionex) for online desalting and then separated on a PepMap C18 RP nano column (3 μm; 75 μm × 15 cm) (Dionex). The eluted peptides were detected in the LTQ Orbitrap Velos through a nano ion source with a 10-μm analyte emitter (New Objective). Data were acquired with Xcalibur 2.1 software (Thermo Fisher Scientific).

The MS/MS raw spectra from iTRAQ experiments were processed with Proteome Discoverer 1.1 (Thermo Fisher Scientific), and a subsequent database search was performed using Mascot Deamon version 2.2.04 (Matrix Science) with a T. ni protein sequence database containing 15,536 sequence entries generated by combining 12,457 sequences (including 12,294 ESTs) downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) on November 20, 2009, and 3,079 sequences (including 2,992 ESTs) generated in our laboratory. For the iTRAQ quantitative analysis and protein identification, peptide mass tolerance and fragment mass tolerance values were 10 ppm and 30 mDa, respectively. The significance threshold was set at a 95% confidence interval, and only those peptides passing this filter were used for protein identification. Furthermore, proteins identified as containing at least two peptides with a P value < 0.001 determined by Mascot probability analysis were analyzed further. Intensities of the reporter ions (114, 115, 116, and 117) from iTRAQ tags on fragmentation were used for quantification, and the relative protein ratios were normalized at the median ratio for the fourplex in each set of experiments.

Internal errors for the iTRAQ analysis were examined with the two technical replicates for each BBMV protein sample included in the iTRAQ runs to ensure the quality of the analysis. The internal error was defined such that for 95% of the identified proteins, the difference in protein ratio between the two technical replicates [│log2(117/115)│ and │log2(116/114)│] was below this value (31). For the two sample sets analyzed in this study, the internal errors of the iTRAQ analyses were 0.25 and 0.27 (log2 ratio), respectively. Similarly, biological variation was determined with the data from two independently prepared sets of midgut BBMV samples [Δlog2 ratio = │log2 ratio(sample set 1) − log2 ratio(sample set 2)│]. The significance cutoff point was set such that the variations of 95% of the identified proteins between the two sample sets was below this value (31).

SDS/PAGE Analysis of BBMV Proteins and Protein Identification by Nano LC-MS/MS.

BBMV proteins (50 μg) were separated by 10% SDS/PAGE and then stained with Coomassie brilliant blue R250. Selected protein bands were excised and subjected to in-gel digestion with trypsin for nano LC-MS/MS analysis with the LTQ Orbitrap Velos or the Synapt HDMS MS system at Cornell University's Proteomics and Mass Spectrometry Core Facility. MS/MS data analysis for protein identification was performed as described above.

Toxin Overlay Binding Assay and Western Blot Analysis.

Toxin overlay binding assays of Cry1Ac to T. ni larval midgut BBMV proteins were conducted as described by Bravo et al. (32). The binding of Cry1Ac to the BBMV proteins on the membrane blot was detected by probing with a Cry1Ac-specific rabbit antibody, followed by the secondary anti-rabbit IgG antibody conjugated with alkaline phosphatase, and then visualized with a colorimetric reaction with nitroblue tetrazolium/bromochloroindolyl phosphate. Duplicate BBMV protein samples from the susceptible and resistant strains were included in the SDS/PAGE, and the gel was stained with Coomassie brilliant blue R250. The protein bands matching the positive bands from the toxin overlay binding assay were excised for protein identification by nano LC-MS/MS analysis as described above.

For Western blot analysis of APN1 in T. ni larvae, proteins from midgut tissue or midgut BBMV proteins were separated by SDS/PAGE and transferred onto Immobilon-P membrane (Millipore). The membrane was treated in 5% nonfat milk and 0.5% Tween-20 and then incubated with a polyclonal antibody specific to T. ni APN1 at 4 °C overnight, followed by incubation with the alkaline phosphatase–conjugated secondary antibodies after several washes of the membrane with PBS. Positive antibody reactions were visualized by a colorimetric reaction as described above.

qRT-PCR.

Total RNA was isolated from individual midguts of fifth instar larvae using the Qiagen RNeasy Mini Kit coupled with an on-column digestion procedure with DNase, following the manufacturer's instructions, and then used for cDNA synthesis with the Promega ImProm-II reverse-transcription system. The cDNA preparations from individual larvae were used for qRT-PCR analysis. Real-time PCR samples were prepared in iQ SYBR Green Supermix (Bio-Rad), and reactions were carried out using the iQ5 RT-PCR detection system (Bio-Rad). qRT-PCR included an initial hot start at 95 °C for 3 min, followed by 40 cycles of amplification at 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s. Relative transcript levels of six T. ni APN genes in the test larvae compared with the control Cornell strain were calculated using the ΔΔCT method, with an actin gene transcript as an internal control for sample normalization.

Genetic Linkage Analyses.

For linkage analysis of APN1 and APN6 genes with Cry1Ac resistance, a backcross family between the GLEN-Cry1Ac-BCS strain and the Benzon strain was prepared by a single-pair cross of a female F1 progeny with a male from the GLEN-Cry1Ac-BCS strain. Individuals from the backcross family were reared on an artificial diet (non–Cry1Ac-selected) or treated with 500 μg/mL of Cry1Ac for 7 d as described by Wang et al. (16) to select Cry1Ac-resistant individuals (Cry1Ac-selected). Larval genomic DNA was prepared from individual larvae using a rapid DNA isolation method (33). A 665-bp genomic DNA fragment for an APN1 gene and a 406-bp genomic DNA fragment for an APN6 gene were amplified from each larva by PCR and subsequently sequenced to determine the allele types.

For linkage analysis of APN1 and APN6 gene transcript levels with Cry1Ac resistance, a single-pair cross was prepared between a male from GLEN-Cry1Ac-BCS and a female from its near-isogenic Cornell strain to generate F1 progeny. An F1 female was backcrossed with a male from the GLEN-Cry1Ac-BCS strain, and an F1 male was backcrossed with a female from the GLEN-Cry1Ac-BCS strain to generate two backcross families, known as backcross family a and backcross family b, respectively. The progenies from each backcross family were divided into two groups with and without selection with 500 μg/mL of Cry1Ac toxin as described above. Fifth instar larvae from the Cry1Ac-selected and -nonselected groups were dissected to isolate the midgut tissue, and the midgut tissue was processed for Western blot analysis of APN1 protein and qRT-PCR analysis for APN gene transcription as described above.

Acknowledgments

We thank Wendy Kain for excellent technical assistance, Sheng Zhang for critical advice and help with the proteomic analysis, and Xin Zhang for the antibodies used in this study. This study was supported by Agriculture and Food Research Initiative Competitive Grant 2008-35302-18806 from the US Department of Agriculture's National Institute of Food and Agriculture.

Footnotes

The authors declare no conflict of interest.

Data deposition: The DNA sequences reported in this paper have been deposited in the GenBank database (accession nos. JF303656JF303663).

*This Direct Submission article had a prearranged editor.

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