Single amino acid mutation in an ATP-binding cassettetransporter gene causes resistance to Bt toxin Cry1Abin the silkworm, Bombyx moriShogo Atsumia,1,2, Kazuhisa Miyamotoa,1, Kimiko Yamamotob, Junko Narukawab, Sawako Kawaia, Hideki Sezutsuc,Isao Kobayashic, Keiro Uchinoc, Toshiki Tamurac, Kazuei Mitab, Keiko Kadono-Okudab, Sanae Wadaa, Kohzo Kandad,Marian R. Goldsmithe, and Hiroaki Nodaa,3

aDivision of Insect Sciences, bAgrogenomics Research Center, and cGenetically Modified Organism Research Center, National Institute of AgrobiologicalSciences, Tsukuba 305-8634, Japan; dFaculty of Agriculture, Saga University, Saga 840-8502, Japan; and eBiological Sciences Department, University of RhodeIsland, Kingston, RI 02881

Edited by Fred L. Gould, North Carolina State University, Raleigh, NC, and approved April 26, 2012 (received for review December 15, 2011)

Bt toxins derived from the arthropod bacterial pathogen Bacillusthuringiensis are widely used for insect control as insecticides orin transgenic crops. Bt resistance has been found in field popula-tions of several lepidopteran pests and in laboratory strains se-lected with Bt toxin. Widespread planting of crops expressing Bttoxins has raised concerns about the potential increase of resis-tance mutations in targeted insects. By using Bombyx mori as amodel, we identified a candidate gene for a recessive form of re-sistance to Cry1Ab toxin on chromosome 15 by positional cloning.BGIBMGA007792-93, which encodes an ATP-binding cassette trans-porter similar to human multidrug resistance protein 4 and orthol-ogous to genes associated with recessive resistance to Cry1Ac inHeliothis virescens and two other lepidopteran species, was ex-pressed in the midgut. Sequences of 10 susceptible and seven re-sistant silkworm strains revealed a common tyrosine insertion in anouter loop of the predicted transmembrane structure of resistantalleles. We confirmed the role of this ATP-binding cassette trans-porter gene in Bt resistance by converting a resistant silkwormstrain into a susceptible one by using germline transformation. Thisstudy represents a direct demonstration of Bt resistance gene func-tion in insects with the use of transgenesis.

genome | map-based cloning | toxin binding | linkage analysis | piggyBac

The bacterial pathogen, Bacillus thuringiensis, produces in-secticidal proteins that are used as selective orally ingested

insecticides. The genes of the insecticidal toxin are also in-troduced into Bt-resistant crops. Increasing use of the toxins hasthreatened to increase the prevalence of Bt resistance in insectpest populations since its first discovery in 1985 in Plodia inter-punctella (1). A key problem in agricultural production is how toavoid the development of Bt-resistant pest populations (2, 3).A number of Bt-resistance mechanisms have been reported,

including mutations in cadherin and aminopeptidase genes (4).Themost common type of resistance is “mode I,” characterized byrecessive inheritance, high resistance level, and reduced bindingof toxin to a putative midgut receptor (5). Some lepidopteranpests, e.g., Plutella xylostella and Heliothis virescens, show charac-teristics of mode I resistance. However, Bt resistance is not fullyexplained by these mutations, and themolecular basis for this typeof resistance has not been unequivocally established in these pestspecies (6). Elucidation of Bt-resistance genes, especially thoseinvolved in the resistance of major pest populations, is of greatimportance for understanding the detailed mode of action andextending the practical application of these environmentallysafe molecules.Recently, a mutation in a class of ATP-binding cassette (ABC)

transporters was proposed to be associated with Bt resistance ina laboratory population of H. virescens (7). This study used theBombyx mori genetic map (8, 9) and genome sequence, aided bythe report of chromosomal linkage analysis of a Bombyx Bt

resistance gene (10) and a high level of chromosome synteny be-tween these two species. Although mutations in the orthologousABC transporters (ABCC2) were reported to be associated withBt resistance in Trichoplusia ni and P. xylostella (11), without directfunctional assays of themechanism of resistance, the evidence thatthis ABC transporter is involved in Bt resistance of these pestsremains circumstantial. This raises two important research issues.One is to confirm that mutation of the ABC transporter geneABCC2 is causally related to Bt resistance, and the second is toexplore the function of this gene in the resistance mechanism.This paper reports direct evidence that Bt resistance is caused

by a mutation in an orthologous ABC transporter in B. mori byintroducing a Bt-susceptible allele into a resistant silkworm byusing transgenesis. That a positional cloning study to seek the Btresistance gene in B. mori was performed independently from theH. virescens study (7) using available Bombyx genome informationclearly confirms this gene (ABCC2) is the causal agent of Bt re-sistance, albeit with a different form of the toxin. Further, inB.mori, resistance in the transporter gene seems to be attributableto a single tyrosine insertion in an outer loop of the predictedtransmembrane protein, an unexpectedly dramatic effect thatpromises to yield new insights into the function of this protein.

ResultsInsecticidal Screening of Silkworm Strains Using Cry1Ab. We testedsusceptibility to Cry1Ab toxin in 133 inbred silkworm strains andfound a wide concentration range required for lethality. We chosetwo strains in which the median lethal concentration (LC50) ofnewly hatched larvae differed by 315 fold, Chinese no. 2 (C2; re-sistant; LC50 of 0.5664 μg protein/cm2) and Ringetsu (Rin; sus-ceptible; LC50 of 0.0018 μg protein/cm2). Both strains weresusceptible to Cry1Aa toxin (C2, LC50 of 0.0310 μg protein/cm;Rin, LC50 of 0.0122 μg protein/cm2), indicating no cross-resistancebetween Cry1Ab and Cry1Aa. Cry1Ab resistance was previously

Author contributions: S.A., K. Miyamoto, K.Y., H.S., K.K.-O., M.R.G., and H.N. designedresearch; S.A., K. Miyamoto, J.N., S.K., H.S., and H.N. performed research; K.Y., I.K., K.U.,T.T., K. Mita, and K.K. contributed new reagents/analytic tools; S.A., K. Miyamoto, H.S.,S.W., and H.N. analyzed data; and M.R.G. and H.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in GenBank(accession nos. AB620070–AB620075, AB621548, and JQ774504).1S.A. and K. Miyamoto contributed equally to this work.2Present address: Ishihara Sangyo Kaisha, Central Research Institute, Kusatsu 525-0025,Japan.

3To whom correspondence should be addressed. E-mail: hnada@affrc.go.jp.

See Author Summary on page 9674 (volume 109, number 25).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1120698109/-/DCSupplemental.

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mapped as a single recessive trait to linkage group 15 (chromo-some 15) among 28 linkage groups by using restriction fragmentlength polymorphisms (10).

Linkage Analysis Using SNP Markers. We initiated map-based clon-ing of the resistance locus by using the two aforementioned strains,C2 and Rin, based on the completed silkworm genome sequence(12–14) and an integrated physical-genetic map (8, 9). Takingadvantage of the lack of chromosomal crossing over in females, wefirst confirmed the linkage assignment of the resistance trait bySNP marker-based analysis (8) by using surviving progeny froma backcross (BC1) between an F1 female (C2 female × Rin male)and a C2male. The yield of BC1 survivors at a preliminary-defineddose (0.031 μg protein/cm2) expected to kill 100% of susceptiblelarvae was 48.9%, consistent with resistance being under thecontrol of a single recessive gene (Table 1). We extracted andamplified DNA from 19 surviving fifth-instar larvae by using pri-mers corresponding to the genome region previously shown tohave SNP(s) in the two strains, C2 and Rin (8), and determinedgenotypes (C2/C2 or C2/Rin) by sequencing the PCR products (SIAppendix, Fig. S1). All the surviving larvae were homozygous (C2/C2) only for chromosome 15 (SI Appendix, Table S1), confirmingthat the Bt-resistance trait was located on chromosome 15.

Comparison with Known Bt Resistance Genes. To date, several geneshave been implicated in Bt resistance in lepidopteran pests andin the nematode, Caenorhabditis elegans. To ascertain whetherthe strain C2 resistance gene corresponded to any of these po-tential candidates, we examined their chromosome assignmentsin the silkworm by using KAIKObase (http://sgp.dna.affrc.go.jp/KAIKObase/) (15). Glycosyltransferase genes of B. mori werePCR-amplified, cloned, and sequenced by using newly designedprimers (Bre primers in SI Appendix, Table S2). None of thegenes for cadherin-like peptide (16), aminopeptidases (17, 18),glycosyltransferases (Bre-2–5) (19, 20), alkaline phosphatase (21),chlorophyllide-binding protein (22), α-amylase (23), or MAPKp38 (24) were located on chromosome 15 (SI Appendix, TableS3), indicating the presence of a different form of Bt resistancein strain C2.

Protoxin Activation and Toxin Binding. Gut protease is required toactivate Bt protoxin, and lack of major gut proteases is associatedwith a form of toxin resistance (25, 26); conversely, high enzymaticactivity may quickly digest toxin, resulting in low susceptibility.Therefore, we compared midgut enzyme activity between strainsC2 and Rin. Gut enzyme extracts from both strains digestedCry1Ab protoxin protein (130 kDa) to the active toxin proteinform (60 kDa) with no marked differences in the protoxin di-gestion profiles (SI Appendix, Fig. S2), indicating that the re-sistance in C2 was not related to the gut enzyme digestion process.We examined binding of toxins to the midgut brush border

membrane vesicles (BBMVs) in susceptible Rin and resistant C2strains under the hypothesis that recessive resistance might be re-lated to a defect in midgut receptors that bind Bt toxins. We in-cubated biotinylated Cry1Ab toxin with BBMVs prepared from thetwo silkworm strains and determined the amount of toxin bound to

the BBMVs in competition with excess unlabeled toxin by usinga streptavidin-peroxidase chemiluminescence detection system.Cry1Ab specifically bound to BBMV of both susceptible Rin andresistant C2 strains (SI Appendix, Fig. S3). We detected no visibledifference between the two strains, indicating that initial Cry1Abbinding to midgut receptor(s) occurred to an equivalent extent.

Map-Based Cloning. We carried out map-based cloning for theresistance gene on chromosome 15 in three stages by usingprogeny from male informative backcrosses (BC1) in single-pairmatings between a C2 female and an F1 (C2 female × Rin male)male. We first determined a broad candidate region for the re-sistance locus by using 44 larvae that survived toxin treatment ata discriminating dose (>0.031 μg protein/cm2) using 17 SNPmarkers on chromosome 15. As before, we expected the sur-viving larvae to be homozygous for resistance (C2/C2) and het-erozygous larvae (C2/Rin) to be susceptible. We determined thehomozygous or heterozygous state for all SNP marker regions bydirect sequencing of PCR products (SI Appendix, Table S4). Thehomozygous region among all 44 samples was localized betweenmarkers 15–016 and 15–089 on chromosome 15, which we esti-mated at 11.4 cM in the genetic map (Fig. 1).To narrow down the location of the Bt-resistance mutation, we

performed two more rounds of mapping experiments. Weobtained 400 new DNA samples from resistant larvae of the maleinformative backcross generation and sequenced the PCR prod-ucts from two SNP-PCR markers, 15–016 and 15–089. We soughtsamples that showed homozygosity for one marker and hetero-zygosity for the other, indicating a crossover had occurred be-tween these two primer sites in one of the sister chromosomes. Ina second round of mapping (SI Appendix, Table S5), we used 10PCR-SNPmarkers on 32 larvae to narrow the candidate region toapproximately 1.0 cM, which corresponded to a physical distanceof approximately 1 Mb located between SNP markers 15–011 and15–089 (Fig. 1). In a third round of mapping (SI Appendix, TableS6), we screened another set of 1,365 resistant backcross larvae;from these, we selected 15 samples that were homozygous in onemarker region (15–011 or 15–089) and heterozygous in another.By using 17 PCR-SNP markers, including 16 newly designed ones(SI Appendix, Table S2), we finally narrowed the candidate regionto ∼82 kb between markers 15–327-4 and 15–218 (Fig. 1).

Determination of Candidate Gene. Six genes—BGIBMGA007735,007793, 007736, 007792, 007791, and 007737—were predicted inthe 82-kb candidate region by genemodels in KAIKObase version2.1.0 (Fig. 1 and Table 2) (15); the marker 15–327-4 was locatedinside the predicted gene, 007735. Of these six genes, we found007735, 007793, 007736, and 007792were expressed in themidgutof C2 and Rin by RT-PCR, excluding 007791 and 007737 ascandidates (Fig. 2). Determination of the cDNA sequences of thefour expressed genes revealed that 007792 and 007793 belongedto a single gene and 007736 was present in the intron region of007792–93. We concluded that 007736 was annotated incorrectly,as its PCR product seemed to correspond to an immature mRNA.Consequently, we predicted two bona fide candidate genes in thenarrowed region, 007735 and 007792–93, both of which showedhomology to members of the ABC transporter superfamily.The nucleotide sequences of 007735 were identical between the

two silkworm strains in the region inside the critical SNP markers(GenBank accession no. AB621548), suggesting this gene was un-likely to be responsible for Bt resistance. In contrast, the sequencesof 007792–93 were significantly different between C2 and Rin (SIAppendix, Fig. S4; accession nos. AB620074 and AB620075, re-spectively): 39 different nucleotides in the protein coding regionyielded 13 different amino acid residues, including one insertion/deletion. From these results we concluded that 007792–93 was themost plausible candidate gene for the Bt resistance and furtherexamined its expression in 11 silkworm organs and tissues by RT-

Table 1. Bioassay of Cry1Ab toxin in two strains

Race/cross No. tested No. survived Survival, %

C2 60 60 100Ringetsu 60 0 0F1: C2 × Ringetsu 60 0 0BC1: (C2 × Ringetsu) × C2 135 66 48.9

For each assay, 15 first instar larvae were reared on a 2 × 4-cm mulberryleaf disk applied with 0.031 μg/cm2 of Cry1Ab protoxin. A fresh leaf wasprovided after 2 d and surviving larvae were recorded after 4 d.

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PCR. We found that 007792–93 was expressed in the midgut, butnot in the fat body, silk glands, Malpighian tubules, hemocytes,testis, ovary, or integument (SI Appendix, Fig. S5), a pattern con-sistent with a role in Bt toxin action.We determined the nucleotide sequences of gene 007792–93

in six additional Bt-resistant and nine susceptible silkworm strains(SI Appendix, Table S7) to determine which sequence differencesin the coding region were responsible for Bt resistance. Strainsthat showed dominance for resistance in the original toxin surveyand preliminary genetic studies were excluded. Although thesequence comparison among the 17 strains revealed manypolymorphisms, only one showed a fixed difference betweenresistant and susceptible strains (Fig. 3 and SI Appendix, Fig. S6):the insertion of three consecutive nucleotides encoding tyrosinein the 007792–93 gene product in resistant strains. The presenceof this common polymorphism in a predicted ABC transporterexpressed in larval midgut strongly implicated this gene in con-tributing to Bt resistance.

Introduction of Susceptible Gene into Resistant Strain. To confirmthat 007792–93 was the causative agent of the Cry1Ab resistance,we introduced a copy of the gene from susceptible strain Rin (Rin-007792–93) into a resistant strain. The recipient resistant strain

was the nondiapausing white-eyed silkworm strain (w1-pnd), de-rived from another resistant strain (w1-c) used routinely fortransgene expression (27). We established two transgenic strains(SS16-1 and SS16-3) expressingRin-007792–93 under an upstreamactivating sequence (UAS) together with EGFP as a selectablemarker. Southern blot analysis and inverse PCR of genomic DNArevealed that SS16-1 had two copies of the transgenes, on chro-mosomes 15 and 23, and SS16-3 had one copy, on chromosome 25(SI Appendix, Fig. S7). We crossed these males with females ofa previously established GAL4 driver strain carrying DsRed2 (52–2) (28) and selected offspring that possessed both Gal4 and Rin-007792–93 by examining eye colors derived from DsRed2 andEGFP at a late embryonic stage (SI Appendix, Fig. S8).We tested the resistance levels of the parental and transgenic

silkworms at the second and fourth larval instars by feedingCry1Ab toxin on mulberry leaf disks and recording mortality after4 d. We first examined the Bt toxin response of parent (Rin andC2), recipient (w1-c and w1-pnd), and GAL4-driver (52–2) strainsat second instar (Table 3). The susceptible strain, Rin, had an LC50of 0.006 μg toxin/cm2, in contrast with the LC50 of the resistantstrain, C2, which was greater than 17.6 μg toxin/cm2. The recipientand driver strains had LC50 values of 1.9 to 22 μg toxin/cm2, also in

15-016

15-089

42 cM 11.4 cM 1.0 cM / 1 Mb 82 kb

15-015

15-075

15-034

15-062

15-027

15-006

15-04115-095

15-011

15-050

15-015

15-062

15-011

15-089

15-01115-327-415-916

15-427-915-427-215-21815-31115-308

15-304

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15-20215-215

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15-221

15-218

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15-427-2BGIBMGA007791

BGIBMGA007793BGIBMGA007736BGIBMGA007792

BGIBMGA00773515-016

15-08920.119.1

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3.7

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BGIBMGA007737

1st 2nd 3rd

Fig. 1. The process of mapping the Bt resistance gene on chromosome 15. Three rounds of mapping analyses were performed by using SNP markers on 44,32, and 15 Bt-resistant BC1 larvae for the first, second, and third mapping screens. Homozygosity (C2/C2) or heterozygosity (C2/Rin) for each marker site wasdetermined by sequencing PCR products. Markers in magenta were used as boundaries for the subsequent mapping round or gene prediction after the threescreens. Six genes were predicted in the final 82-kb region in KAIKObase.

Table 2. Genes in the 82-kb region on chromosome 15 predicted in KAIKObase

Gene name Strand Position Size, bp Exon size, bp Description

BGIBMGA007735 + 8912489–8944193 31,705 3,807 ABC transporterBGIBMGA007793 — 8949057–8952178 3,122 999 ABC transporterBGIBMGA007736 + 8952469–8952919 451 229 UndefinedBGIBMGA007792 — 8956687–8966706 10,020 2,150 ABC transporterBGIBMGA007791 — 8969410–8981067 11,658 5,418 UndefinedBGIBMGA007737 + 8992602–8992829 228 228 Undefined

Six genes were predicted. cDNA and genome sequence analyses indicated that BGIBMGA007793 and 007792were parts of the same gene and BGIBMGA007736 was located in one of the intron regions of the gene.Therefore, four genes, BGIBMGA007735, 7792–93, 007791, and 007737 were actually predicted. + representsforward sequence and — reverse sequence in KAIKObase. bp, base pairs.

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a resistant range.We then tested the two transgenic strains, SS16-1and SS16-3, at two larval stages. The LC50 values of second-instarlarvae from crosses between 52–2 and SS16-1 or SS16-3 were0.0054 and 0.0033 μg toxin/cm2, respectively (Table 3 and SIAppendix, Fig. S9), showing susceptibility to Bt toxin. As controls,offspring from crosses between w1-c females and the UAS-trans-genic strains lacking a GAL4 driver showed high resistance totoxin (LC50 values 48.7 and >800 in SS16-1 and SS16-3, re-spectively). Crosses between the 52–2 GAL4 driver strain and theoriginal w1-c strain also produced resistant offspring (LC50 valueof 3.9). We obtained similar results for fourth instar larvae, con-firming that introducing Rin-007792–93 into Bt-resistant silkwormstrains made them highly susceptible to Cry1Ab toxin (Table 3).

Expression of Introduced Gene in Transgenic Silkworms. We con-firmed expression of the introduced gene into the transgenicsilkworms by real-time RT-PCR. As the transgenic silkwormspossessed a pair of endogenous 007792–93 genes, we used primersdesigned for the 3′ region, which included mismatched nucleo-tides for distinguishing the expression of the endogenous andexogenous genes separately (SI Appendix, Fig. S10 and Table S2).We successfully quantified expression of the genes in the midgutof fourth-instar C2 and Rin larvae (Fig. 4A). We also quantifiedexpression in three groups of transgenic animals: 52–2 × SS16(GAL4×UAS), w1-c × SS16 (no-GAL4×UAS), and 52–2×w1-c(GAL4 × no-UAS) by real-time RT-PCR using the two effectorstrains, SS16-1 (Fig. 4B) and SS16-3 (Fig. 4C). The exogenousRin-007792–93 gene(s) was highly expressed in 52–2 × SS16-1 and 52–2 × SS16-3 (GAL4 × UAS; Fig. 4 B and C, a). Although we couldnot compare directly the expression levels of the introduced gene,

Rin-007792–93, and the endogenous gene, w1-c-007792–93, be-cause of different PCR efficiency with the use of different primers,the expression level of Rin-007792–93 was apparently as high asthat of w1-c-007792–93 in SS16-1 and SS16-3. Notably, Rin-007792–93 was expressed at a low level even in the absence ofGAL4 (Fig. 4 B and C, b), indicating leaky expression of the in-troduced gene. However, the effect of the leaky expression on theresistance level was unclear, as resistance of transformed insects inthe absence of aGAL4 driver (w1-c× SS16-1 or× SS16-3; Table 3)was higher than in untransformed controls (w1-c, Table 3).

Structure of ABC Transporter Gene. The gene 007792–93 showedhigh homology to human ABC transporter gene ABCC4, which isknown to be involved in multidrug resistance (SI Appendix, Fig.S11). Two ABC domains were predicted in the silkworm protein,including Walker A motif, Walker B motif, and C-motif. Twotransmembrane domains each consisting of six transmembraneregions (TMs) were also predicted (SI Appendix, Fig. S11). Theinsertion of tyrosine was predicted in or on the edge of the secondouter loop between TM 3 and TM 4 (Fig. 5 and SI Appendix,Fig. S12).

DiscussionGermline introduction of a functional form of a gene associatedwith resistance to a Bt toxin converted an insect from resistance tosusceptibility, confirming a central role for the 007792–93 gene inCry1Ab toxin action. The achievement of the cloning and confir-mation of the function of the cloned gene was accomplished byusing three main research platforms. First, the success of the map-based cloning was much the result of a well-maintained genomedatabase, KAIKObase (http://sgp.dna.affrc.go.jp/KAIKObase/)(15) and a high density of SNP markers on the genetic map (http://sgp.dna.affrc.go.jp/LinkageMap/cgi-bin/index.cgi) (8, 9). Second,the transformation technique, first developed in the silkwormamong lepidopteran insects (29), clearly demonstrated that thecandidate 007792–93 gene played a key role in the Bt toxin re-sponse. Finally, selection of two suitable Bt resistant/susceptiblestrains for map-based cloning and determination of the site of themutation were made possible by the large number of naturallyresistant and susceptible silkworm strains maintained in the Ge-netic Resource Center of the National Institute of AgrobiologicalSciences (http://www.gene.affrc.go.jp/databases_en.php?section=animal). Successful use of these resources further validates thesilkworm as an effective research model for lepidopteran insects.Further, that the function of this gene appears to have been con-served in Lepidoptera belonging to widely divergent superfamilies(Bombycoidea, Noctuoidea, Yponomeutoidea) supports the valueof comparative studies between B. mori and other members of thislarge and highly pestiferous insect clade.To confirm the ABC transporter gene was responsible for the

resistance/susceptibility to Cry1Ab toxin, we introduced a sus-ceptible Rin allele into a resistant strain, w1-pnd, and performedall subsequent crosses to activate and test the function of thetransgene with resistant strains. We did not perform the trans-genesis in the reverse direction, introducing a resistant allele ofthe gene from the C2 strain into a susceptible strain, becauseresistance behaved as a recessive trait in heterozygotes with oneresistant and one susceptible allele, and the introduction of a re-sistant allele of 007792–93 into a strain carrying two endogenoussusceptible alleles would not be expected to alter the phenotype.Sequence analyses of seven independent resistant and 10 sus-

ceptible strains suggested that only a single common amino acid(codon) insertion/deletion was responsible for the change infunction of the Bombyx Bt resistance gene 007792–93. This genepossesses domains required for the functions of an ABC trans-porter (SI Appendix, Fig. S11) and shows high homology to thehuman multidrug resistance gene ABCC4 (30). An orthologuewas recently reported as a candidate for Cry1Ac resistance in H.

M 1 2 3 4 5 6

Rin

C2

Fig. 2. Expression of the six predicted genes in the midgut. RT-PCR productsof BGIBMGA007735, 007793, 007736, 007792, 007791, and 007737 (marked1–6) are shown from midgut of Rin and C2. BGIBMGA007735–007792(marked 1–4) show PCR products of expected size in both Rin and C2. M,DNA marker.

J1_RKi_RBe_RC2_RC7_RCsek_RN15_RYosh_SBag_SN65_SEu12_SAnn_SCamM_SMy_SPMy_SRin_Se21_S

Fig. 3. Sequence alignment of putative amino acids deduced from a por-tion of gene 007792–93 (from residues 223–246 in C2; SI Appendix, Figs. S4and S6). Seven Bt resistant strains (Upper) and 10 susceptible strains (Lower)are shown. Tyrosine is present in the resistant strains and lacking in thesusceptible strains.

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virescens and named ABCC2 (7). However, its function in Bt re-sistance is still unclear. Two plausible alternative Bt resistancemechanisms may be considered. One is that the protein is in-volved in binding and/or insertion of Bt toxin into the midgutmembrane, working as a receptor in a mechanism similar to thoseproposed for a cadherin-like protein (16) or aminopeptidases(31), or as a membrane channel (7), and the insertion of tyrosinein the second loop outside the membranemay interfere with theseprocesses. Another possibility is that the ABC transporter worksto detoxify the Bt protein by excluding it from cells in a manneranalogous to that used by members of ABC transporter subfamilyC in drug resistance (32). However, the second mechanism is ir-reconcilable with the finding that resistance associated with thisgene is a recessive trait in three evolutionarily disparate lepi-dopteran species besides B. mori (7, 11). If the ABC transporteracts in the detoxification of Cry toxins, resistance should bedominant, because detoxification would be expected to occur in

heterozygous (i.e., R/S) insects, including silkworm. On the con-trary, if the ABC transporter is involved with toxin binding ormembrane insertion, both alleles in the sister chromosomesshould carry a mutation (i.e., be homozygous) for resistance.Gahan et al. (7) recently reported that a frameshift mutation

in an ABC transporter of H. virescens, which is orthologous tosilkworm gene 007792–93 (ABCC2) and located in a syntenicchromosome region, is linked genetically with resistance toCry1Ac. The H. virescens mutation is accompanied by reducedbinding of Cry1Ac and Cry1Ab toxins to midgut membranes.This suggests the possibility that the exposed loop region wherethe tyrosine insertion occurred in B. mori is a toxin-binding re-gion (Fig. 5). However, Cry1Ab bound equally to the BBMVfrom susceptible and resistant silkworm strains (SI Appendix, Fig.S3). As Cry1A toxins are shown to bind a cadherin-like protein(33) and aminopeptidase N (34) in B. mori, the failure to finda difference in the BBMV toxin binding assay between the two

Table 3. Susceptibility to Cry1Ab toxin in transgenic silkworms

Strains cross, female × male No. testedLC50, μg

protein/cm2 95% CL Slope ± SE*

Original strains, second instarRingetsu (Rin) 168 0.00616 0.0027–0.0147 1.85 ± 0.27Chinese 2 (C2) 168 >17.6 — —

w1-c 168 1.94 1.13–3.54 1.10 ± 0.16w1-pnd 144 22.1 10.8–123 1.23 ± 0.3352–2 168 12.7 2.56–37300 0.74 ± 0.15

Gal4 × UAS, second instar52–2 × SS16-1 144 0.00543 0.0040–0.0074 3.62 ± 0.76w1-c × SS16-1 144 48.7 19.4–4230 1.33 ± 0.4852–2 × SS16-3 168 0.00329 0.0001–0.0024 1.38 ± 0.19w1-c × SS16-3 144 846 — —

52–2 × w1-c 144 3.89 2.01–9.69 0.89 ± 0.17Gal4 × UAS, fourth instar

52–2 × SS16-3 144 0.00942 0.0067–0.0129 4.67 ± 1.03w1-c × SS16-3 108 131 20.1–11.0 × 107 0.69 ± 0.2752–2 × w1-c 90 2.36 1.01–7.12 0.87 ± 0.21

CL, confidence limit; LC50 = median lethal concentration. w1-c, diapausing recipient strain used for maintaining the transgenicstrains; w1-pnd, nondiapausing recipient strain in which the susceptible gene (Rin-007792–93) was introduced; 52–2, GAL4 driverstrain with DsRed2; SS16-1 (2 copies) or SS16-3 (1 copy), transgenic strains expressing EGFP and Rin-007792–93. We tested susceptibilityto Cry1Ab toxin at second instar for SS16-1 and SS16-3 and at fourth instar for SS16-3. We tested individual larvae by providing a leafapplied with Bt toxin in 24-well plates at second instar or six-well plates at fourth instar. We fed a fresh leaf after 1 d and recorded thenumber of surviving larvae after 4 d. — means that confidence limits were not generated because of low mortality rates.*Slope calculated by probit analysis.

Fig. 4. Expression of introduced and endogenous 007792–93 genes in silkworms. Susceptible (Rin-007792–93) and resistant (C2- or w1-c-007792–93) geneswere individually detected in midguts of fourth-instar larvae by real-time RT-PCR. Expression levels relative to those of a ribosomal protein gene (RpL32) areshown with SEs. The number of larvae tested is shown above the columns. Closed and open boxes indicate susceptible and resistant genes, respectively. Theasterisk indicates no expression. (A) Parental strains, Rin and C2; expression of endogenous genes, Rin-007792–93 and C2-007792–93, respectively, using a real-time PCR method that differentially detects susceptible and resistant genes. (B and C) Gene expression level in offspring of SS16-1 and SS16-3; expression ofthe exogenous (Rin-007792–93) and endogenous (w1-c-007792–93) genes is shown in closed and open boxes. a, offspring from 52–2 × SS16; b, offspring fromw1-c × SS16; c, offspring from 52–2 × w1-c; offspring from 52–2 × SS16 showing expression of the introduced susceptible gene as well as the endogenousgene. Leaky expression of the susceptible gene is observed in the offspring from w1-c × SS16.

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strains indicates that further experiments are needed to clarify themechanism underlying the present form of Bt toxin resistance.Studies on the impact of other amino acid variants on the degreeof resistance or susceptibility among silkworm strains may helpidentify additional critical regions of the 007792–93 protein andelucidate their roles in Bt toxin action.The origin of the resistant allele carrying a tyrosine insertion is

unclear. The predicted sequence of the ABC transporter inBombyx mandarina (accession no. JQ774504), the nearest wildancestor to the silkworm, has only 3 aa differences from suscep-tible and resistant alleles of the B. mori gene reported here, andcarries a deletion of the tyrosine in question (SI Appendix, Fig.S13). Hence, B. mandarina is expected to be susceptible to Bttoxin. Although B. thuringiensismay be present on mulberry leavesroutinely fed to laboratory and commercial strains, rearing ofdomestic silkworms takes place in a relatively controlled and hy-gienic environment. The majority of susceptible strains were ofEuropean and tropical origin, whereas resistancewas foundmostlyin traditional and genetically improved Chinese and Japanesestrains. This suggests that, after the resistant allele arose, it hasbeen maintained as a cryptic mutation for long periods of timeunder nonselective conditions. Preliminary phylogenetic analysisbased on the ABC transporter nucleotide sequences indicates thatresistant strains belong to a single clade; however, additionalvariants are needed for a well-supported evolutionary scenario.

Materials and MethodsSilkworm Strains Used. Two B. mori strains, C2 (resistant to Cry1Ab toxin, race401; http://www.gene.affrc.go.jp/ex-nises/bombygen/indexJ4-eng.html) andRin (susceptible to Cry1Ab toxin, race 606; http://www.gene.affrc.go.jp/ex-nises/bombygen/indexJ6-eng.html), were used for map-based cloning; theywere reared on mulberry leaves or artificial diet (Nosan) at room tempera-ture. The strains used for transgenesis were reared on artificial diet. Theyincluded the recipient, w1-pnd, a white eye color and nondiapausing mutantstrain of diapausing strain w1-c, a GAL4 driver strain, 52–2, which expressesthe GAL4 protein in the midgut and DsRed2 in the eyes (28, 35), and SS16-1and SS16-3, two newly established UAS-Rin-007792–93 strains expressingEGFP. B. mori strains are listed in SI Appendix, Table S7.

Insecticidal Screening by Cry1Ab Toxin. The Cry1Ab toxin from B. thuringiensissubsp. kurstakiHD-1was expressed in Escherichia coli (36). Bacteria expressingthe toxin were centrifuged and protoxin inclusions were recovered by themethod of Lee et al. (37). The protein content of the suspension of protoxininclusions was estimated by a Lowry assay (38) using BSA (Wako PureChemical) as a standard. The protoxin content in the suspension was esti-mated by a modified method of Brussock and Currier (39). The protoxin waseluted by using 7% (wt/vol) SDS/PAGE, and the content of 130 kDa proteinwas measured by image analyzing software (Quantity One; Bio-Rad).

For Bt toxin screening, a mulberry leaf fragment (2 × 4 cm) coated with 80μL of diluted suspension of Cry1Ab protoxin was fed to 15 newly hatchedlarvae for 2 d. The dose (usually >0.03 μg/cm2) was determined to be highenough to kill 100% neonate larvae of susceptible strains that were ho-mozygous or heterozygous for susceptible genes, but not to kill those ho-mozygous for resistance (Table 1). Larvae were fed fresh mulberry leaves for2 d after toxin exposure, and mortality was recorded 4 d after the initial

application of Bt toxin. Transgenic silkworms were tested individually atsecond or fourth instar on treated leaf discs under similar conditions in 24-well or six-well plastic plates. Probit analysis was carried out by using SPSSsoftware (version 7.5.1J; SPSS) to determine LC50.

Toxin Digestion by Gut Enzymes. Cry1Ab toxin inclusion expressed in E. coliwasincubated in 0.1M Na2CO3/NaHCO3 (pH 10.2) with 10 mM DTT for 1 h on ice.After centrifugation for 20 min at 5,000 × g, the supernatant was passedthrough a 0.45-μm filter and dialyzed overnight at 4 °C, followed by pre-cipitation with ammonium sulfate and washing with sterile distilled water.Prestarved fifth-instar larvae were frozen and thawed, the midgut was dis-sected, and the liquid that leached out of the midgut was collected. Themidgut juicewasmixedwith the Cry1Abprotoxin. After incubationat 25 °C for0 min, 1 min, 10 min, or 120 min, the solution was inactivated with heat andanalyzed with SDS/PAGE (e-PAGEL; ATTO) followed by staining with Coo-massie brilliant blueR250. The toxinmoleculeswere also visualized byWesternblot analysis. Proteins in the PAGEgelwere transferred onto PVDFmembranes(Hybond-P; GE Healthcare) by Transblot SD (Bio-Rad). After blockingwith skimmilk, the membranewas incubated with rabbit antiserum against Cry1A toxin(40), then with peroxidase-labeled anti-rabbit antibody (GE Healthcare), andvisualized using a HistMark TrueBlue Peroxidase System (KPL).

Toxin Binding. Protoxin (130 kDa) prepared as described earlier was activatedwith trypsin (41), and the activated toxin (60 kDa) was labeled with biotinusing an ImmunoProbe Biotinylation Kit (Sigma). Midgut BBMV were pre-pared from C2 and Rin strains as described by Wolfersberger et al. (42). TheBBMV suspension in 0.01 M PBS solution (including 0.15 M NaCl) was storedat −80 °C until use, and evaluated by aminopeptidase activity (31). BBMV (25μg protein) was incubated in PBS solution with 0.1 μg of biotin-labeledCry1Ab for 1 h at 25 °C. Excess unlabeled Cry1Ab (1 or 10 μg) was also addedfor the competition assay for toxin binding. The BBMVs were collected bycentrifugation (10,000 × g, 10 min) and washed three times with PBS solu-tion at 4 °C, followed by electrophoresis (e-PAGEL) and staining with an EzStain Silver Kit (ATTO). Proteins were blotted onto PVDF membranes(Hybond-P), which were incubated with a streptavidin-horseradish peroxi-dase conjugate (GE Healthcare) for 1 h at 25 °C. The biotin-labeled toxin wasdetected with ECL Plus (GE Healthcare) by using a Lumino Imaging AnalyzerFAS-1000 (TOYOBO).

Cloning and Sequencing.Glycosyltransferasegeneswereclonedandsequencedusing total RNAof theC2 strain. cDNAwas synthesizedby using SUPERSCRIPT II(Invitrogen)andanoligo(dT) primer. ThecDNAwasamplifiedwithPCRprimers(SI Appendix, Table S2) and 3′-RACEwas performed. PCR productswere clonedinto a pGEM-T vector (Promega). DNAamplified from the clones by colony PCRwas used for sequencing reactions. The sequence analysis was performedwithan ABI Prism 3730 sequencer using a BigDye Terminator kit (Applied Bio-systems). Cloning and sequencing of other genes in this study were carried outin a similar manner. PCR products amplified with SNP-PCR primers were di-rectly sequenced (without cloning) using the SNP-PCR primers from both ends.

Linkage Analysis and Positional Cloning of the Resistance Gene. SNP-basedlinkage analysis and recombination mapping were performed by PCR ampli-fication of the SNP region and sequencing the PCR products (8, 9). GenomicDNAwas isolated from an anterior leg of adult moths of grandparental strains(C2 female and Rin male) and parental F1 individuals by using DNAzol (Invi-trogen). For the BC1 generation, genomic DNAwas isolated from legs of fifth-instar larvae. Nineteen segregant BC1 larvae that survived after screeningwithCry1Ab were used for the linkage analysis. Thirty SNP markers were used, in-cluding three markers for chromosome 15 and a single marker for each of theother 27 chromosomes (9). The PCR products were directly sequenced byBigDye terminator cycle sequencing (Applied Biosystems) by using the samePCR primers. The homozygous (A) or heterozygous (H) state of each linkagegroup was determined (SI Appendix, Fig. S1). A pair of sister chromosomes foreach of the 28 linkage groups should be composed of the two same chro-mosomes that originated from C2, or different chromosomes from C2 and Rin.Larvae that possessed a pair of homozygous sister chromosomes should showresistance to Bt toxin if the resistance gene was located on this chromosome.Therefore, the chromosome (i.e., linkage group) carrying the resistance traitshould be homozygous in all resistant larval samples examined.

To determine the locus of the resistance gene on chromosome 15, F1 males(C2/Rin) were crossed with C2 females (C2/C2). As chromosomal crossing-over(i.e., recombination) occurs in silkworm males but not in females (43, 44),reciprocal backcrosses were used for chromosome linkage assignment andpositional cloning. Recombination between sister chromosomes was used tofind the homozygous region in chromosome 15 of Bt-resistant BC1 larvae. In

NH2

COOH

ABCABCIntracellular space

Extracellular space

Fig. 5. Schematic structure of the ABC transporter. Twelve transmembranedomainswere predicted basedon the aminoacid sequenceofBGIBMGA007792-93 (SI Appendix, Fig. S11) using TMHMM version 2.0. The tyrosine residue(magenta dot) was predicted to be located on the second outer loop (SIAppendix, Fig. S12).

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addition to already known SNP markers (PCR primers) on chromosome 15,new SNP-PCR primers that could distinguish C2 and Rin were designed aftersequencing the corresponding region of the two strains.

Transgenesis. An established silkworm GAL4/UAS system (27) was used fortransgenesis. Two piggyBac vector constructs were used: a driver construct(GAL4 line) BmA3-0052–2 (52–2) containing Gal4 and DsRed2 genes that wasused previously (28, 35), and a new effector construct (UAS line) containinga Rin-007792–93 gene and an EGFP gene (SI Appendix, Fig. S8A). The codingsequence of the Rin-007792–93 gene was amplified from cDNA from themidgut of Rin by using primers with an Xba I cutting site (SI Appendix, TableS2) and cloned into a pGEM-T vector. The insert DNA was digested with Xba Iand subcloned downstream of the GAL4 binding site of UAS (Bln I site) ofthe plasmid pBacMCS[UAS-3×P3-EGFP] (45). The insert sequence of the re-sultant effector vector was confirmed by DNA sequencing.

Transgenesis was performed as described previously (27, 29). The eye-colormutant strain, w1-pnd, a nondiapausing mutant of diapausing strain w1-c,was used as recipient. Two EGFP-positive UAS lines (SS16-1 and SS16-3) wereestablished and maintained by crossing with w1-c. Females of 52–2, whichexpresses the GAL4 protein in the midgut and DsRed2 in the eyes, werecrossed with males of the UAS lines (SS16-1 and SS16-3). The DsRed- andEGFP-positive offspring were selected at a late embryonic stage. As experi-mental controls, offspring from crosses between w1-c females and SS16-1 orSS16-3 males and between 52–2 females and w1-c males were used.

Southern Blot Analysis and Inverse PCR. The copy number of the PiggyBacvector bearing the Rin-007792–93 gene was examined by genomic Southernblot analysis. Genomic DNA was prepared from embryos using a DNeasyBlood and Tissue Kit (Qiagen) or from adults as reported previously (27).Approximately 2 μg each of genomic DNA was digested with Pst I, Hpa I, orEco RV and blotted onto a nylon membrane (Hybond-N; GE Healthcare) afteragarose gel electrophoresis. The EGFP gene fragment (672 bp) amplifiedwith primers KS113 and KS248 (SI Appendix, Table S2) was labeled by usingan AlkPhos direct labeling and detection system (GE Healthcare) and used asa probe. The chromosomal insertion sites of the vector on the chromosomes

were determined by inverse PCR. Two pairs of primers for first and secondrounds of PCR were designed on the left and right arms of the vector (SIAppendix, Table S2). After sequencing the second set of PCR products usingsecond-round primers, a BLAST search of the sequences was carried outagainst the genome sequence in KAIKObase.

Real-Time RT-PCR. To confirm the expression of the exogenous transformedgene, the endogenous and exogenous genes were detected by using primersthat amplified each of the genes separately. Because sequences of both geneswere similar and itwas difficult to design specific primer pairs for theORFs, theprimers were designed in the 3′ region of the genes (SI Appendix, Fig. S9 andTable S2). The primers included mismatched nucleotides with the corre-sponding sequences of cDNA to ensure the differential amplification betweenthe two genes. Both genes in transgenic silkworms were quantified on a real-time thermal cycler (LightCycler 480 Real-Time PCR System; Roche Diag-nostics). The midguts were dissected from fourth-instar larvae, and total RNAwas extracted by using an RNeasy Mini Kit (Qiagen). cDNA was synthesizedfrom the RNA with an oligo(dT) primer by using a PrimeScript RT reagent Kit(Takara Bio) in a 10-μL reaction volume. The reactionmixturewas then diluted10-fold with MilliQ water. Real-time RT-PCR was carried out in 20-μL reactionvolumes containing 5 μL of template cDNA or standard DNA, 0.75× SYBRGreen PCR premix (Roche Diagnostics), and 10 pmol of each primer. PCRconditions were 95 °C for 5min followed by 40 cycles of 95 °C for 10 s, 60 °C for20 s, and 72 °C for 15 s. The absence of undesirable byproducts was confirmedby automated melting curve analysis. The expressed transcript levels werestandardized to that of the ribosomal protein gene RpL32 (AY769302) (46).

ACKNOWLEDGMENTS. We thank S. Kobayashi and M. Kitazume fortechnical support, N. Nakashima for support on the Bt toxin binding assay,E. Kosegawa and O. Ninagi for providing silkworm strains, R. Sato andS. Takeda for valuable suggestions, and A. Papanicolaou for bioinformaticsupport. This work was supported by the Special Research Fund for Symbiosisand Biological Interactions of the National Institute of AgrobiologicalSciences and by the Integrated Research Project for Insects Using GenomeTechnology of the Japanese Ministry of Agriculture, Forestry, and Fisheries.

1. McGaughey WH (1985) Insect resistance to the biological insecticide Bacillus thur-

ingiensis. Science 229:193–195.2. Tabashnik BE, Gassmann AJ, Crowder DW, Carrière Y (2008) Insect resistance to Bt

crops: evidence versus theory. Nat Biotechnol 26:199–202.3. Tabashnik BE, Van Rensburg JBJ, Carrière Y (2009) Field-evolved insect resistance to Bt

crops: definition, theory, and data. J Econ Entomol 102:2011–2025.4. Bravo A, Soberón M (2008) How to cope with insect resistance to Bt toxins? Trends

Biotechnol 26:573–579.5. Heckel DG, et al. (2007) The diversity of Bt resistance genes in species of Lepidoptera. J

Invertebr Pathol 95:192–197.6. Baxter SW, Zhao JZ, Shelton AM, Vogel H, Heckel DG (2008) Genetic mapping of Bt-

toxin binding proteins in a Cry1A-toxin resistant strain of diamondback moth Plutella

xylostella. Insect Biochem Mol Biol 38:125–135.7. Gahan LJ, Pauchet Y, Vogel H, Heckel DG (2010) An ABC transporter mutation is

correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin. PLoS Genet 6:

e1001248.8. Yamamoto K, et al. (2006) Construction of a single nucleotide polymorphism linkage

map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end

sequences. Genetics 173:151–161.9. Yamamoto K, et al. (2008) A BAC-based integrated linkage map of the silkworm

Bombyx mori. Genome Biol 9:R21.10. Hara W, Miyamoto K, Yonsun K, Kanda K (2005) A novel single resistant gene against

BT-toxin in the silkworm Bombyx mori, was located at the molecular genetic map

based on RFLP. Biotechnology of Bacillus thuringiensis,, eds Binh ND, Akhurst RJ,

Dean DH (Science and Technics, Hanoi, Vietnam), Vol. 5, pp 271–276.11. Baxter SW, et al. (2011) Parallel evolution of Bacillus thuringiensis toxin resistance in

lepidoptera. Genetics 189:675–679.12. International Silkworm Genome Consortium (2008) The genome of a lepidopteran

model insect, the silkworm Bombyx mori. Insect Biochem Mol Biol 38:1036–1045.13. Mita K, et al. (2004) The genome sequence of silkworm, Bombyx mori. DNA Res 11:

27–35.14. Xia QY, et al.; Biology Analysis Group (2004) A draft sequence for the genome of the

domesticated silkworm (Bombyx mori). Science 306:1937–1940.15. Shimomura M, et al. (2009) KAIKObase: an integrated silkworm genome database

and data mining tool. BMC Genomics 10:486.16. Vadlamudi RK, Weber E, Ji I, Ji TH, Bulla LA, Jr. (1995) Cloning and expression of

a receptor for an insecticidal toxin of Bacillus thuringiensis. J Biol Chem 270:

5490–5494.17. Crava CM, et al. (2010) Study of the aminopeptidase N gene family in the lep-

idopterans Ostrinia nubilalis (Hübner) and Bombyx mori (L.): Sequences, mapping and

expression. Insect Biochem Mol Biol 40:506–515.

18. Knight PJK, Knowles BH, Ellar DJ (1995) Molecular cloning of an insect aminopepti-

dase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin. J Biol Chem

270:17765–17770.19. Griffitts JS, et al. (2003) Resistance to a bacterial toxin is mediated by removal of

a conserved glycosylation pathway required for toxin-host interactions. J Biol Chem

278:45594–45602.20. Marroquin LD, Elyassnia D, Griffitts JS, Feitelson JS, Aroian RV (2000) Bacillus thur-

ingiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode

Caenorhabditis elegans. Genetics 155:1693–1699.21. Jurat-Fuentes JL, Adang MJ (2004) Characterization of a Cry1Ac-receptor alkaline

phosphatase in susceptible and resistant Heliothis virescens larvae. Eur J Biochem 271:

3127–3135.22. Pandian GN, et al. (2008) Bombyx mori midgut membrane protein P252, which binds

to Bacillus thuringiensis Cry1A, is a chlorophyllide-binding protein, and the resulting

complex has antimicrobial activity. Appl Environ Microbiol 74:1324–1331.23. Fernandez-Luna MT, et al. (2010) An alpha-amylase is a novel receptor for Bacillus

thuringiensis ssp. israelensis Cry4Ba and Cry11Aa toxins in the malaria vector mos-

quito Anopheles albimanus (Diptera: Culicidae). Environ Microbiol 12:746–757.24. Cancino-Rodezno A, et al. (2010) The mitogen-activated protein kinase p38 is in-

volved in insect defense against Cry toxins from Bacillus thuringiensis. Insect Biochem

Mol Biol 40:58–63.25. Lilley M, Ruffell RN, Somerville HJ (1980) Purification of the insecticidal toxin in

crystals of Bacillus thuringiensis. J Gen Microbiol 118:1–11.26. Oppert B, Kramer KJ, Beeman RW, Johnson D, McGaughey WH (1997) Proteinase-

mediated insect resistance to Bacillus thuringiensis toxins. J Biol Chem 272:

23473–23476.27. Imamura M, et al. (2003) Targeted gene expression using the GAL4/UAS system in the

silkworm Bombyx mori. Genetics 165:1329–1340.28. Ito K, et al. (2008) Deletion of a gene encoding an amino acid transporter in the

midgut membrane causes resistance to a Bombyx parvo-like virus. Proc Natl Acad Sci

USA 105:7523–7527.29. Tamura T, et al. (2000) Germline transformation of the silkworm Bombyx mori L.

using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84.30. Schuetz JD, et al. (1999) MRP4: A previously unidentified factor in resistance to nu-

cleoside-based antiviral drugs. Nat Med 5:1048–1051.31. Knight PJK, Crickmore N, Ellar DJ (1994) The receptor for Bacillus thuringiensis CrylA

(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca

sexta is aminopeptidase N. Mol Microbiol 11:429–436.32. Russel FGM, Koenderink JB, Masereeuw R (2008) Multidrug resistance protein 4

(MRP4/ABCC4): A versatile efflux transporter for drugs and signalling molecules.

Trends Pharmacol Sci 29:200–207.

Atsumi et al. PNAS | Published online May 25, 2012 | E1597

AGRICU

LTURA

LSC

IENCE

SPN

ASPL

US

33. Hara H, et al. (2003) A cadherin-like protein functions as a receptor for Bacillusthuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx morilarvae. FEBS Lett 538:29–34.

34. Nakanishi K, et al. (2002) Aminopeptidase N isoforms from the midgut of Bombyxmori and Plutella xylostella — their classification and the factors that determine theirbinding specificity to Bacillus thuringiensis Cry1A toxin. FEBS Lett 519:215–220.

35. Sakudoh T, et al. (2007) Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene. Proc Natl Acad Sci USA 104:8941–8946.

36. Kim YS, Kanda K, Kato F, Murata A (1998) Effect of the carboxyl-terminal protein ofCry1Ab in Bacillus thuringiensis on toxicity against the silkworm, Bombyx mori. ApplEntomol Zool (Jpn) 33:473–477.

37. Lee MK, Milne RE, Ge AZ, Dean DH (1992) Location of a Bombyx mori receptorbinding region on a Bacillus thuringiensis delta-endotoxin. J Biol Chem 267:3115–3121.

38. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with theFolin phenol reagent. J Biol Chem 193:265–275.

39. Brussock SM, Currier TC (1990) Use of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis to qantify Bacillus thuringiensis delta-endotoxins. Analytical

Chemistry of Bacillus thuringiensis, eds Hickele LA, Fitch WL (American ChemicalSociety, Washington, DC), pp 78–87.

40. Shimada N, Miyamoto K, Kanda K, Murata H (2006) Binding of Cry1Ab toxin, a Ba-cillus thuringiensis insecticidal toxin, to proteins of the bovine intestinal epithelialcell: An in vitro study. Appl Entomol Zool (Jpn) 41:295–301.

41. Indrasith L, et al. (1991) Processing of delta endotoxin from Bacillus thuringiensissubspp. Kurstaki HD-1 and HD-73 by immobilized trypsin and chymotrypsin. ApplEntomol Zool (Jpn) 26:485–492.

42. Wolfersberger M, et al. (1987) Preparation and partial characterization of amino acidtransporting brush border membrane vesicles from the larval midgut of the cabbagebutterfly (Pieris brassicae). Comp Biochem Physiol A Physiol (Bethesda) 86:301–308.

43. Sturtevant AH (1915) No crossing over in the female of the silkworm moth. Am Nat49:42–44.

44. Tanaka Y (1913) A study of Mendelian factors in the silkworm, Bombyx mori. J CollAgric Tohoku Imp Univ 5:91–113.

45. Uchino K, et al. (2006) Evaluating promoter sequences for trapping an enhancer ac-tivity in the silkworm Bombyx mori. J Insect Biotechnol Sericol 75:89–97.

46. Shinoda T, Itoyama K (2003) Juvenile hormone acid methyltransferase: A key regu-latory enzyme for insect metamorphosis. Proc Natl Acad Sci USA 100:11986–11991.

E1598 | www.pnas.org/cgi/doi/10.1073/pnas.1120698109 Atsumi et al.