d Tenebrio ty of Tennessee, )areeffective Bt ( ction, Chinese Academy of ), cabbage leaf beetle thology, Universi e Key Laboratory for Biology of Plant Health Research, Manhattan, KS, USA 2011 Society of Chemical Industry c Jeffrey A Fabrick, c Colaphellus Bacillus thuringiensis , Institute of Plant Prote cadherin -based biopesticides. but are not effective in controlling coleopteran pests. Bt and 6 ∗ Crioceris quatuordecimpunctata a Correspondence to: Zhongren Lei, Stat Diseases and Insect Pests USDA ARS Center for Grain and Animal USDA ARS US Arid Land Agricultural Research Center, Maricopa, AZ, USA Institute of Insect Sciences, College of Agriculture andUniversity, Hangzhou, Biotechnology, Zhejiang PR China Agricultural Sciences, Beijing 100193, PR China. E-mail: [email protected] State Key Laboratory for Biology ofPlant Plant Protection, Diseases Chinese and Academy Insect of Pests, Agricultural Sciences, Institute Beijing, of PR China Department of Entomology and Plant Pa Knoxville, TN, USA dherin peptide, rTmCad1p, would enhance Cry3Aa toxicity ecticidal activity has potential for use in increasing range ∗ c a e d b In comparison, commercial Bt-based pesticide productscoleopteran targeting pests areexpressing the based Cry3Aa toxin. on Bt san diego or tenebrionis cauliflower, ). To assess the effect of rTmCad1p on Cry3Aa toxicity, neonate creasing amounts of rTmCad1p. The data demonstrated that Brenda Oppert, 1 b -based pesticides for efficient control of important coleopteran pests. Bacillus thuringiensis -based , which Bt Bt commercial formulations Bt has developed Bt Phaedon brassicae Bt kurstaki Bt and Zhongren Lei synergist; cadherin; Coleoptera Bacillus thuringiensis Crioceris quatuordecim- e has been extended to Bt toxin oligomerization by a polypeptide from the Cry3Aa receptor cadherin in Bt Tenebrio molitor Bt toxins are also expressed in Bt ;Crytoxin; ry1Aa, Cry1Ab, Cry1Ac, Cry2A and 2 Phaedon brassicae by a , ) and daikon leaf beetle ( 3–5 produces one or more crystal (Cry) proteins, Jianhua Gao : 1076–1081 www.soci.org Juan Luis Jurat-Fuentes, Bt a 67 a toxins are effective against lepidopteran pests Bt 2011; crops to provide pest control with reduced chemical Bacillus thuringiensis ) is an environmentally safe biological insecticide. Bt At present, the application of Bt

These (Coleoptera: Tenebrionidae), it was hypothesized that this ca 6 7

2011 Society of Chemical Industry In China, the production and application of Colaphellus bowringi c contains five crystal proteins, C Cry2B. the production of vegetables. However, almost all formulations used in China have been based on increasing from less thanin 0.3 million 2005. kg in 1985 to 30 million kg quickly in the past 20 years, with the use of Pest Manag Sci The spore-forming gram-positive bacterium During sporulation, 1INTRODUCTION Berliner ( which are stored as insoluble crystalline inclusions. BACKGROUND: Biopesticides containing Cry insecticidal proteins from the bacterium Chenxi Liu, Abstract Yulin Gao, pesticides have beento used control worldwide agricultural and forthan forestry more pests, 90% than and of account 60 all years for biopesticides. more Increased toxicity of (wileyonlinelibrary.com) DOI 10.1002/ps.2149 Research Article Received: 11 October 2010 Revised: 4 January 2011Cry3Aa Accepted: against 1 February 2011punctata Published online in Wiley Online Library: 14 April 2011 bowringi on important cruciferous vegetable crops such as cabbage and pesticide applications. transgenic against many lepidopteran pests, but there is a lack of ( Based on the reported increase in molitor towards coleopteran larvae. To test this hypothesis, thechrysomelid relative toxicity vegetable of pests Cry3Aa, with of or China without rTmCad1p, was against evaluated. damaging RESULTS: Cry3Aa toxicity was evaluated in the spotted asparagus beetle ( larvae were fed Cry3Aa toxin alone or in combination with in Cry3Aa toxicity was significantlymortality. increased in all three vegetable pests, resulting inCONCLUSION: as The much application of as rTmCad1p a to 15.3-fold enhance increase Cry3Aa in ins larval

and activity levels against coleopteran pests displaying low susceptibility to fragment Keywords:

1076 1077 ).  was E. coli 600 C. ends of the BL21 (DE3), ◦  (DE3)RP g. 80 gene (accession µ  − 0 and 3 E. coli. . expression vector   cry3Aa gene, the pET28a vector C. When the OD ◦ The approximate 30 kDa cDNA (nucleotides 3963 kanamycin and incubated 1 22 − C and 70% relative humidity , pH 7.4). The Cry3Aa protoxin L ◦ 4 cry3Aa µ 2 strains from laboratory stocks -GGA TCC atg ata aga aag gga  g rTmCad1p peptide andligatedwithalinkerproduced gene was cloned into the pET-28a PO ± µ 2 TmCad1 wileyonlinelibrary.com/journal/ps gene were inoculated in LB (Luria- -F (5 sites located at the 5 250 rpm) at 37 BamHI -GAG CTC tta att cac tgg aat aaa ttc a-3 cry3Aa  Cry3Aa toxin ∼ TransformedArcticExpress cry3Aa T. molitor Bt BamHI SacI and ,2mMKH 22 -R (5 4 and B. thuringiensis XbaI SacI HPO sites for cloning the 2 SacI )and BamHI  C, and newly hatched larvae were used for bioassays. All ◦ and 2 ± Using PCR, 20 were screened with primers targeting the number AY728 479): and recombinant vectors wereclones verified harboring by the sequencing. Selected Bertani) medium containing 50 2.3 Preparation of A 582 bp region from the (Stratagene, La Jolla, CA)production cells of were the usedNJ). peptide for (GenScript The large-batch Corporation, protein (20solubilized L) Piscataway, and was purified by expressed affinity chromatographyconditions, as under hybrid as inclusion previously bodies described. and was (Invitrogen, Carlsbad, CA),rTmCad1pconstruct. was used to generate the pET151- with a 14 : 10 h light : dark photoperiod. 2.2 Preparation of gga aga-3 22.1 MATERIALS AND METHODS Source ofAdult insects SABExperimental were Station (Beijing). obtainedon An asparagus from plants SAB within colony a theEggs controlled was of environmental CLB chamber. maintained and Changping DLB wereAgricultural kindly Asparagus provided Sciences by (Jiangxi, Jiangxi China) Academy of andUniversity Huazhong (Wuhan, Agricultural China) respectively.26 Eggs were incubated at with vigorous shaking ( pests that currently have limitedimportantly, the available use of control rTmCad1p options. as Cry3Aa More the enhancer amount may of reduce toxin needed for effective control, lengthenactivity residual and delay the onset of resistance in target insects. between 0.6 and 1,1 IPTG mM, and was the added cultureCells to was were a incubated harvested final for by three concentrationsonicated additional centrifugation of in hours. ice-cold (12 000 1 g MCell NaCl, for 5 lysates 1 mM min) EDTA were and andprecipitate centrifuged 1% was Triton resuspended (12 000 X-100. in rpm PBS bufferKCl, for (137 10 mM mM 10 NaCl, Na min), 2.7 mM and the amplicons, the amplified (Novagen, San Diego, CA) expression vector. To generate adequate BamHI wasexcisedwith by annealing two oligonucleotides (oligo1:aac ttt cta aag aag gaa gag, ata and oligo2: att gatttt). cct ttg cct The tct ttt taa ligated agt plasmid taa aca was aaa tta transformed into purified peptide wasTris/HCl, pH dialyzed 8.0, 0.01% Triton-X-100) against and concentrationby peptide 10% evaluated SDS-PAGE and densitometry buffer comparisons with known (10 BSA standards. mM Peptide was lyophilized for storage at Using the insects were maintained at 26 was examined byelectrophoresis sodium (SDS-PAGE) and quantified dodecyl by densitometry sulfate–polyacrylamide using a GS-800 calibrated densitometer gel and Quantity One image analysis software from Bio-Radserum Laboratories albumin standards (Hercules, ranging from CA) 0.1 to and 2 bovine to 4548), subcloned into pET151-D-TOPO , , 30 . 12,13 , Both 9 Similar Ingested Cry toxin 28 ,andthe Notably, in Helicoverpa cadherin fragment www.soci.org 10 23 2011 Society of Chemical Industry (Coleoptera: The present ., 13 Aedes aegypti c An alternative larvae. 24 et al 16,17 , 32–34 . Manduca sexta More recently, Chen , corresponding to Alterations of toxin- A generally accepted cadherin receptor for 11 8 15 , . 19,20 . receptor in Binding to these secondary A. gambiae Tenebrio molitor Bt Tenebrio molitor 23 M. sexta H. virescens Diabrotica virgifera virgifera In contrast, different cadherin Colaphellus bowringi Crioceris quatuordecimpunctata To evaluate whether rTmCad1p insecticidal Cry toxins has been 26 and 31 Bombyx mori 29 Bt Phaedon brassicae Another peptide containing the putative Anopheles gambiae cadherin, rTmCad1p, was demonstrated Heliothis virescens larvae. M. sexta. 27 : 1076–1081 and 67 This cadherin fragment also increased Cry3Aa identified the first functional Cry toxin receptor and H. armigera 22 14 22 . Pectinophora gossypiella 25 2011; T. molitor et al T. molitor and enhanced the activity of Cry1A toxins by promoting 18 identified a putative cadherin 12 21 . H. armigera A protein fragment containing the predicted toxin-binding The mode of action of In agreement with the crucial role of cadherin for Cry According to the model proposed by Bravo Enhancement of Cry3Aa activity by a the required activationconfer and, remarkable more pest importantly, specificity binding to steps Cry proteins. model suggests that Cry toxintivates binding intracellular to pathways cadherin leading receptors to ac- cell death. region from to bind Cry3Aaoligomerization. toxin specifically and promote Cry3Aa toxin extensively studied in lepidopteran larvae. insecticidal crystal proteinsproteinases are from the activated digestivethe to peritrophic insect matrix, a gut activated fluids. toxinsproteins toxic on bind After the form to midgut crossing specific microvilli. by receptor Inmidgut lepidopterans, several proteins insect haveSpecifically, Cry1A been receptor proposed functionality hasfor as been demonstrated cadherin Cry proteins toxin from receptors. and Fabrick Cry toxicity enhancement effects wereof cadherin discovered from for fragments armigera toxin oligomerization. Pest Manag Sci Ostrinia nubilalis et al daikon leaf beetle (DLB), can potentiate Cry3Aaexpressed toxicity rTmCad1p in wasrelevant coleopteran field used pests of pests, with vegetablesspotted in heterologously asparagus Cry3Aa China, beetle including in (SAB), the the bioassays cabbage of leaf beetle (CLB), peptides haveagainst been several demonstrated insectBtR to pests. cadherin enhance Forcadherin (CR12-MPED) example, Cry ectodomain from a 12 toxicity and fragmentregion, containing of a critical the toxin-binding data demonstrateto that enhance rTmCad1p Cry3Aa can toxicity be against used several significantly coleopteran field receptors concentrates toxin oligomers ingions specific membrane called re- lipid rafts,pores that where result toxin in cell oligomers death insert by osmotic and shock. form both models, cadherin is a critical contactis point pivotal for for Cry intoxication. toxins that intoxication, some cadherin peptides fed with Crycompete toxins evidently for toxinin binding and cause a reduction in toxicity cadherin from a coleopteran insect, Tenebrionidae), suggesting similarities inCry the toxins across mode insect of taxonomic orders. action of toxin-binding site of an binding to cadherin facilitates further proteolyticessary processing nec- for toxinaffinity oligomerization. for Toxin aminopeptidase oligomers orreceptor display alkaline proteins high phosphatase, attached secondary sylphosphatidylinositol (GPI) to anchor. the cell membrane by a glyco- mode of actionof for protoxin Cry activation, toxins specific binding describes and the cell sequential toxicity. steps binding motifs within lepidopteran cadherin genes are genetically linked to Cry1Ac resistance in toxicity in Cry4Ba potentiated Cry4Ba toxicity in . et al values 50 : 1076–1081 67 05). . 0 2011; < P Pest Manag Sci respectively. In all panels, PBS buffer control 1 − gmL µ 5 . rTmCad1p were not toxic. Mortality was scored after 1 − rTmCad1p enhances Cry3Aa toxicity against neonate larvae of gmL µ and 10 96 h of exposure. Different lettersdifferences above between the means error (chi-square bars analysis, indicate significant Figure 1. SAB (A), CLB (B) and DLB (C). Inwere panels 1.5, A, 0.2 B and and C, 0 the Cry3Aa concentrations significantly different fromincreased Cry3Aa controls. toxicity against Similarly, CLB larvaethe (Fig. rTmCad1p 1 1B). : 5 In also Cry3Aa this : case, rTmCad1p(62%), ratio which resulted in was the highest(8%). significantly mortality Even though greater Cry3Aa toxin than caused relativelyfor with higher DLB mortality Cry3Aa larvae alone (more1 than : 5 20%) toxin compared : peptide with80%. ratio other These significantly species, data increased indicated a toxicity that to rTmCad1pCry3Aa almost significantly toxicity enhanced against larvae oflevels SAB, of enhancement CLB observed, 1 and : DLB. 5 was Based selectedmass as on ratio the optimum the of Cry3Aa : rTmCad1p for further testing. 3.2 Estimationby of rTmCad1p Cry3Aa toxicity enhancement In orderenhancement to effect accurately of rTmCad1pbioassays quantify in were the SAB, performed CLB to specific and calculate Cry3Aa DLB the larvae, Cry3Aa toxicity LC 35 34 37 )for www.soci.org Y Gao 50 2011 Society of Chemical Industry c values were calculated by EPA 50 and LC 36 05. . 0 for SAB, CLB and DLB respectively) or in combination < C, 70% relative humidity and a 14 : 10 h light : dark 1 P ◦ − 2 ± gmL µ To test the effect of rTmCad1p on Cry3Aa toxicity towards Assays for neonate CLB and DLB larvae were conducted on fresh Assays for neonate SAB larvae were conducted on fresh 5–8 cm 5 . with increasing mass ratios of1 : Cry3Aa 5 : and rTmCad1p, 1 including : 50. 1 Fromratio : these 1, assays, for the Cry3Aa optimum Cry3Aa toxicity : enhancement rTmCad1p wasfor determined all to be species. 1 This :effect 5 ratio of was rTmCad1p used on in the bioassays 50% to lethal quantify concentration the (LC wileyonlinelibrary.com/journal/ps 3RESULTS 3.1 rTmCad1plarvae enhances Cry3Aa toxicity to SAB, CLB and DLB To evaluateincreasing the amounts of Cry3Aa rTmCad1p weretoxin potentiating combined concentration causing with 10–20% activity a Cry3Aa larvalon species). mortality of As (depending shown in Fig. rTmCad1p, 1A,Cry3Aa rTmCad1p significantly enhanced toxicity against SABSpecifically, larvae 1 : 1 at and lowas 1 toxin opposed : : 5 peptide to mass 16% ratios. ratiosexposure mortality resulted with of in Cry3Aa SAB 80% alone. mortality, Mortality larvae from to rTmCad1p peptide alone was not The enhancement factor,concentration of defined Cry3Aa asCry3Aa alone mixed the to with ratio cadherin theof peptide, the of lethal was combinations. concentration Non-overlapping the determinedwere 95% for of lethal used confidence each intervals to establishwas significance. Pairwise performed chi-square to analysis dose–response analyze bioassay the usingbioassays effect log comparing a of transformed fixed concentration dose the ofratios toxin data. of enhancer with rTmCad1p, For different in pairwise chi-squareand the mortalities analysis between was treatments were performed, considered significantly different if Briefly, cabbage leavescut were into rinsed the appropriate withwere then size water, completely (6 submersed air cm into differentCry3Aa diameter concentrations dried of toxin circle). and for The 10 s, pieces disk air and cushioned dried with and a placed wet filter onto to a maintain 9 humidity. cm diameter SAB, CLB and DLBexposed larvae, to neonate a larvae0 single of Cry3Aa each species concentration were alone (1.5, 0.2 and Briefly, the plant section wasvertically dipped for to 10 allow s excess inrack the solution in solution, to a held fume drip exhaust hood off, toa placed air 9 dry in cm for diameter 1 a h plastic drying andball. petri then dish placed containing into a moistened cotton leaf disks of Chinese cabbage using a modified leaf method. 2.4 Insect bioassays Each bioassay was conducted with 20 neonateand larvae per replicate three replicatesdetermined after per 96 h concentration. ofif exposure; they Larval larvae did were not mortality considered respondat dead to was contact. 26 All bioassays were performed 2.5 Data analysis Mortality values were correctedAbbott’s for background formula, mortality using each species. photoperiod. Controls included larvae exposed to7.4) buffer or (PBS, rTmCad1p pH alone. long sections of asparagus spears, using a spear dipping method. probit analysis using POLO-PC (LeOra Software, Berkeley, CA).

1078 1079 05) . 0 < P An alternative 41 . standard error of the ± H. armigera reatment only and with Cry3Aa plus wileyonlinelibrary.com/journal/ps Therefore, the peptide shows potential for 31 However, the correlation between toxin enhancement Determinationofthe Cry3Aatoxicity enhancementby rTmCad1p 40 Cry toxin oligomerization was reported as a critical factor for Cry toxicity. development into a new and important tool forin pest agriculture. management results from aasterisk bioassay denotes with a 60between larvae significant larval mortality difference per with Cry3Aa (chi-square concentration. t analysis, InrTmCad1p for panels, overlapping toxin an doses. have been observed. Figure 2. in neonate larvae of SABtoxin with (A), or without CLB fivefold (B) molar and excesslarvae rTmCad1p DLB were in (C). fed to Suspensions neonate a ofexposure. Cry3Aa spear/leaf Each dip data point bioassay. represents Mortality the mean was scored after 96 h of andtoxinoligomerizationhasbeeninconsistent,asatoxin-binding cadherin fragment thatdemonstrated to induced reduce toxicity Cry1Ac in oligomerization was explanation for the observed Cry toxicity enhancement is that 1 − 60 29,38 b = .When gmL n 1 µ cadherin − factor cadherin fragment www.soci.org 33 2011 Society of Chemical Industry . this cadherin Enhancement c gmL 30 with Cry3Aa and µ values for Cry3Aa M. sexta 50 82 50 . 0.38) 0.39) 0.28) 0.42) 4.3 0.39) 7.9 0.29) 15.3 SE) ± ± ± ± ± ± ± Slope Furthermore, increased ( of 3 was reported to enhance 22 50 Tenebrio molitor -resistant lepidopteran pest Bt ) a 1 values of 1.11 and 1 − larvae. Even though a polypeptide 50 50 30 LC with Cry3Aa alone/LC gmL D. virgifera µ (95% CL) 50 ( LC = : 1076–1081 67 may be involved in the observed Cry3Aa toxicity CLB 0.14 (0.08–0.20) 2.98 ( SAB 0.25 (0.19–0.31) 2.39 ( DLB 0.31 (0.23–0.38) 2.84 ( 22 ., 2011; c and coleopteran Previous studies have demonstrated enhancement of Effect of rTmCad1p on Cry3Aa toxicity to neonate SAB, CLB TmCad1 cadherin containing at least one Cry3Aa et al 39 + + + 28,30 , concentration causing 50% mortality; 95% CL, 95% confidence 50 rTmCad1p rTmCad1p rTmCad1p Enhancement factor LC A 1 : 5 mass ratio of Cry3Aa toxin : rTmCad1p peptide was used for all Several peptides based on putative cadherin receptors have Cry3AaCry3Aa DLB 1.33 (0.73–1.90) 2.96 ( Cry3AaCry3Aa CLB 1.11 (0.61–1.59) 2.96 ( limits. b c a TreatmentCry3Aa Insect Cry3Aa SAB 3.82 (3.07–4.62) 2.44 ( and DLB larvae Table 1. Cry3Aa concentrations tested against alllarvae per three concentration). insect species ( rTmCad1p. Enhancement of Cry3Aa activity by a Cry3Aa was combined with fivefold mass excessdose–response of rTmCad1p, curves the shifted to those of(Fig. the lowest 2), Cry3Aa providing doses further evidence for the enhancementtoxicity of by Cry3Aa rTmCad1p. When comparing the LC Pest Manag Sci dipteran 4The DISCUSSION present dataCry3Aa are toxicity the against firstT. chrysomelid report molitor larvae on by thebinding site. a enhancement This peptide finding of supportsCry from recent toxicity reports enhancement demonstrating byinsect cadherins. polypeptide Furthermore, an fragments optimum molar from ratiopeptide of host was toxin identified to that results intoxicity the enhancement highest against levels of three Cry3Aa vegetable important pests. species of beetle been reported to enhance Cry toxicity against lepidopteran, for eachsusceptible species to Cry3Aa, (Table with 1). LC respectively CLB (Fig. 2). andless In DLB susceptible, with comparison, larvae an SAB estimated were LC larvae most were slightly peptides. enhancement, as this mechanism also hasenhancement been of proposed Cry1 for toxins the in Lepidoptera by Cry toxins by rTmCad1plarvae and against increase coleopteran in toxicity and to lepidopteran a based on a cadherintoxicity from of Cry3 toxins towards coleopteran pests, toxin alone or in combination with rTmCad1p,enhancement the highest was toxicity detected forwhile SAB lower but larvae significant (more effects werelarvae than detected (7.9- for 15-fold), and CLB 4.3-fold and respectively). DLB has not yet beenreceptor. demonstrated In to contrast, represent thebased protein a on a fragment functional functional used Cry3 Cry3Aa receptor. in this work is Cry3Aa oligomerization in the presence ofin rTmCad1p, Fabrick as reported , , . in ,A Ba- (Bt) et al et al et al J Appl crystal strains :63–71 et al Plutella Bacillus Toxicon Biochem cadherin (Hubner) :635–644 24 Cell Mol Life 98 Biochemistry Biochemistry Bombyx mori : 1076–1081 , Monitoring JBiolChem . :33–40 (2005). 67 :857–860 (2001). ´ on M and Gill SS, 35 et al ´ on M, Bravo A, :948–954 (2005). Bacillusthuringiensis Bacillus thuringiensis Bacillus thuringiensis 293 Bacillus thuringiensis Helicoverpa armigera Biochim Biophys Acta 2011; Cry1Ab binding and 71 Bacillus thuringiensis Nat Biotechnol Bacillus thuringiensis Proc Natl Acad Sci USA cadherin is a functional subsp. israelensis. lipid rafts are involved in Bacillus thuringiensis Ostrinia nubilalis J Econ Entomol Science Heliothis virescens Bacillus thuringiensis . Cry3Aa toxin. :255–281 (2007). Heliothis virescens iver L, Ellers-Kirk C, Higginson D, S2 cells functions as a receptor 71 Cry1Ab toxin depends on specific :13 863–13 872 (2002). :1407–1416 (2005). Pest Manag Sci 12 -endotoxin of 277 δ Insect Biochem Mol Biol :311–316 (2001). :641–653 (2008). toxins in Cry1A, but not Cry1Fa toxins. toxins kill insect midgut cells? :28 051–28 056 (2004). Manduca sexta 20 ´ on M, Mode of action of 29 Bacillus thuringiensis Tenebrio molitor Drosophila Appl Environ Microbiol Cry1Ac toxicity against ´ . omez I, Pullikuth AK, Sober :29–34 (2003). in pink bollworm. Heliothisvirescens 279 Bacillus thuringiensis and , Association between resistance to Bt cotton and 538 JBiolChem cadherin serves as a putative receptor of the Bacillus thuringiensis Microbiol Mol Biol R et al Crop Prot toxins and biological control. :441–446 (2007). Cell Death Differ Bacillus thuringiensis (L.). J Environ Biol 131 JBiolChem FEBS Lett :1337–1349 (2009). :191–200 (2009). :38–46 (2004). , Three cadherin alleles associated with resistance to , Oligomerization triggers binding of a :5004–5009 (2003). :18 401–18 410 (2009). ´ Bacillus thuringiensis on M, Gill SS and Bravo A, Signaling versus punching hole: how Bacillus thuringiensis 66 Bacillus thuringiensis :423–435 (2007). :9688–9695 (2006). :14 299–14 305 (2004). 424 thuringiensis (2006). transgeniccrop:andenvironmentfriendlyinsect-pestmanagement strategy. of resistance for theCry1Aa and diamondback Cry1Ab toxins moth and to Entomol a Bt commercial formulation. and commercial formulations toxylostella the diamondback moth, 15 Sci Cry and Cyt toxins and their potential for insect control. do toxin activity. toxicity of binding of the toxininsect to cells. the cadherin receptor BT-R1 expressed in for 45 HevCaLP protein mediates bindingof specificity of the43 Cry1A class with Bt resistance in cadherin-likeproteinfunctionsasareceptorfor Cry1Aa and Cry1Ac toxins on midgut epitheliallarvae. cells of protein expressed in repeat 12 mediates toxicity. Indentification, cloning and expressionceptor of from a Cry1Ab(Lepidoptera: European Crambidae). cadherin re- corn borer, with resistance to Cry1Ac Cry1A toxin bindingpore to formation. the midgut epithelium and subsequent Helicoverpa armigera Bacillus thuringiensis 100 Unnithan GC, cadherin genotype in Pink bollworm. Heliothis virescens et al cillus thuringiensis (2005). Aedes aegypti Cry11Aa toxin from 1667 J Fuentes JL, A novel Cry1Ab pore-forming toxin to aminopeptidaseto N insertion receptor leading into membrane microdomains. 284 et al receptor for 4 Romeis J, Meissle M and Bigler F, Transgenic crops expressing 5 Kumar S, Chandra A and Pandey KC, The 6 Wang L, Li XF, Zhang J, Zhao JZ, Wu QJ, Xu B, 7 Mohan M and Gujar GT, Toxicity of 8 Bravo A, Gill SS and Sober 9Sober 10 Pigott CR and Ellar DJ, Role of receptors in 11 Hura H, Atsumi S, Yaoi K, Nakanishi K, Higurashi S, Miura N, 13ZhangX,CandasM,GrikoNB,Rose-YoungLandBullaLA,Jr,Cyto- 14 Flannagan RD, Yu CG, Mathis JP, Meyer TE, Shi X, Siqueira HA, 16 Jurat-Fuentes JL, Gahan LG, Gould FL, Heckel DJ and Adang MJ, The 17 Gahan LJ, Gould F and Heckel DG, Identification of a gene associated 12 Hua G, Jurat-Fuentes JL and Adang MJ, Bt-R1a extracellular cadherin 15 Jurat-Fuentes JL and Adang MJ, The 18 Xu X, Yu L and Wu Y, Disruption of a cadherin gene associated 25 Liu CX, Wu KM, Wu YD, Gao YL, Ning CM and Oppert B, Reduction of 20 Tabashnik BE, Biggs RW, Higginson DM, Henderson S, Unnithan DC, 19 Morin S, Biggs RW, Sisterson MS, Shr 24 Zhuang M, Oltean DI, G 21 Chen J, Aimanova KG, Fernandez LE, Bravo A, Sober 22 Fabrick J, Oppert C, Lorenzen MD, Morris K, Oppert B and Jurat- 23 Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez J, Miranda R, : www.soci.org Y Gao BBMV in Although 22 2011 Society of Chemical Industry c T.molitor Bacillus thuringiensis Bacillus thuringiensis Recent Pat Biotechnol This discovery provides :323–342 (2006). 31 41 -toxin-based biopesticides or Bt This hypothesis is consistent with 27 toxicity represent an opportunity to by rTmCad1p. In the case of rTmCad1p, increased :5–81 (2002). Process Biochem values for Cry3Aa with rTmCad1p were Bt 42 47 50 events remain to be determined. in vivo :28–36 (2009). an environmentally3 friendly alternative. based biopesticides. downstream processing and formulations of and social consequences of the deployment ofAnnu bt Rev transgenic plants. Entomol Considering that coleopterans are some of the most damaging Pectinophoragossypiella 2 Brar S, Verma M, Tyagi RD and Valero JR, Recent advances in 1 Rosas-Garcia NM, Biopesticide production from 3 Shelton AM, Zhao JZ and Roush RT, Economic, ecological, food safety, a recently proposed model,toxin whereby monomers low-affinity to binding binding sites ofis on Cry the brush crucial border epithelium for toxicity. oligomerization was observed in the presence of improve currentlyeffective available control agents commercial againstdemonstrates diverse products the pests. potential for into The use of present aan more work cadherin-based enhancer peptide for Cry3Aa-based as products in controlling chrysomelid pests. similar for all three testeddifferences species. in This cadherin observation binding suggests may that explainto diverse susceptibility Cry3Aa in SABis compared with needed CLB to and determineCry3Aa DLB. the toxicity Further enhancement specific research by rTmCad1p. mechanism responsible for pests ofurgent many need agricultural toinsects. and develop Enhancers effective forestry of biopesticides crops, against there these is an wileyonlinelibrary.com/journal/ps REFERENCES ACKNOWLEDGEMENTS The authors are grateful to ZR LiuSciences), (Jiangxi Academy XP of Wang Agricultural and LZsity) Chen for (Huazhong Agricultural kindly Univer- providingZhang the (IPP) and eggs ZC of Shenbioassays. (ZJU) CLB This for and research their was valuable DLB. supported by suggestion We thethe with earmarked thank Modern fund Agro-industry for J Technology Research(Nycytx-35-gw27) System and of by a China Technology Maturation GrantUniversity from the of Tennessee Research Foundationtrade to JLJF. names Mention or of commercial productsfor in the this purpose publication of isimply providing solely recommendation or specific endorsement by information the US andAgriculture. Department of does not 5CONCLUSIONS The present data supportwith the Cry3Aa use of toxinThe rTmCad1p for observation in that rTmCad1p control combination can reduce of therequiredforefficacysuggeststhatselectionpressurebyCry3Aawill amount coleopteran of Cry3Aa vegetable pests. be reduced and correlates with the reductionin in Cry1Ac resistance cadherin peptides may bindand attract to Cry toxin the molecules, increasing microvillae theinteraction probability on of with toxin midgut receptors. cells susceptibility to Cry3Aa wasenhancement, not the correlated LC to the fold levels of a novel strategy to enhanceresistance insecticidal in activity coleopteran pests and to delaytransgenic insect crops. solution, but

1080 1081 ´ an C, Peptides ,Anen- Manduca Manduca ´ . on M and Biotechnol Spodoptera Helicoverpa ). et al JBiolChem Cry1Ab toxin Cry1Ab toxin toxins against and Cry1A toxins by Plant Health Prog ´ on M, Enhancement Manduca sexta ´ omez I, Gill SS, Sober strain with insecticidal activity Colaphellus bowringi Bacillus thuringiensis Bacillus thuringiensis Bacillus thuringiensis and Bacillus thuringiensis :718–724 (2010). wileyonlinelibrary.com/journal/ps :1097–1103 (2009). 56 0818-01-RS (2006). 65 :583–588 (2009). . Cambridge University Press, London, UK ˜ noz-Garay C, G 30 reduction of Cry1Ac toxicity in :265–267 (1925). Helicoverpa zea, Agrotis ipsilon 18 , Domain II Loop 3 of in vivo Peptides ´ ´ omez I, Gill SS, Bravo A and Sober omez I, Arenas I, Saab-Rincon G, Rodriguez-Almaz Bacillus thuringiensis J Insect Physiol . ´ arez N, Mu Probit Analysis Pest Manag Sci et al :697–703 (2009). aminopeptidase-N and cadherin receptors. . (Lepidoptera: Sphingidae) cadherin fragments function as 31 :32 750–32 757 (2009). :318–323 (2008). ´ enez-Ju Leptinotarsa decemlineata of insecticidal activityfragments of of aformation. toxin-binding cadherin correlates with oligomer J Econ Entomol is necessary to29 induce insect death in mediated is involved insexta a ‘ping pong’284 binding mechanism with gineered against scarabaeidae (Anomala( corpulenta) and Chrysomelidae efficacy of acetamiprid on asparagus beetle. Bravo A, The pre-pore from DOI:10.1094/PHP-2006- (1971). sexta synergists for Cry1A and Cry1C noctuid moths exigua armigera Lett Gill SS, 34 Yan GX, Song FP, Shu CL, Liu JJ, Liu CQ, Huang DF, 36 Abbott WS, A method of computing the effectiveness of an insecticide. 37 Finny DJ, 39 Pacheco S, G 41 Liu CX, Gao YL, Ning CM, Wu KM, Oppert B and Guo YY, Antisera- 35 Kuhar TP, Doughty HB, Hitchner E and Chapman A, Toxicity and field 40 Jim 42 Pacheco S, G 38 Abdullah MAF, Moussa S, Taylor MD and Adang MJ, Bio- Acta cadherin cadherin fragment www.soci.org 2011 Society of Chemical Industry J Insect Physiol c Insect Molec Biol . :1033–1040(2010). Anopheles gambiae Bacillus thuringiensis Chin Agricl Sci Bull bind specifically to a 85 al activity by facilitating toxin- Cry3Aa and Cry3Bb toxicities :13 901–13 906 (2007). proximal extracellular domain Helicoverpa armigera Tenebrio molitor :3086–3092 (2009). (Scopolic). 75 104 : involvment of a cadherin in the rick JA and Oppert C, Novel cadherin Bacillus thuringiensis Baly (Coleoptera: Chrysomelinae). toxins by a fragment of a toxin binding (Balsamo) Vuillemin isolate to daikon leaf Bacillus thuringienssis ApplMicrobiolBiotechnol : 1076–1081 :2582–2588 (2005). Bacillus thuringiensis Manduca sexta 67 25 :5101–5110 (2008). and a fragment of AgCad1 synergizes toxicity. 47 Proc Natl Acad Sci USA Appl Environ Microbiol 2011; Phaedon brassicae Beauveria bassiana ,Cry1Atoxinsof Bacillus thuringiensis :686–693 (2009). :1025–1036 (2002). :338–339 (2006). 55 by a soluble toxin-binding cadherin fragment. entomopathogeneicity of region adjacent to theof membrane- BT-R(1) in 32 et al cadherin. of Ecologica Sin beetle, receptor peptide60/988,919. for Filed with potentiating US PTO 18 Bt November (2008). biopesticides.of Serial a No. hancement of to coleopteran larvaecadherin. by a toxin-binding fragment of an insect fragment enhances Cry1Ac insecticid oligomerformation. cadherin AgCad1 binds theisraelensis Cry4Ba toxin of chemistry study on theCrioceris occurrence quatuordecimpunctata regularity22 and control techniques of 26 Dorsch JA, Candas M, Griko NB, Maaty WS, Midboe EG, Vadlamudi RK, 27 ChenJ,HuaG,Jurat-FuentesJL,AbdullahMAandAdangMJ,Synergism 32 He YR, Lu LH, Kuang ZB, Feng X, Chen HY and Wu YJ, Pathogenicity 28 Hua G, Zhang R, Abdullah MA and Adang MJ, 30 Park Y, Abdullah AF, Taylor MD, Rahman K and Adang MJ, En- 29 Peng D, Xu X, Ye W, Yu Z and Sun M, 33 Zhang JL, Wang ZJ, Yang JG, Shi GR, Xue MY and Jin XH, Preliminary 31 Oppert BS, Jurat-Fuentes JL, Fab Enhancement of Cry3Aa activity by a Pest Manag Sci