Cry1Fa insecticidal protein was successfully radiolabeled with 125I-Na. Specific binding to brush border membrane vesicles was shown for the lepidopteran species Ostrinia nubilalis, Spodoptera frugiperda, Spodoptera exigua, Helicoverpa armigera, Heliothis virescens, and Plutella xylostella. Homologous competition assays were performed to obtain equilibrium binding parameters (Kd [dissociation constant] and Rt [concentration of binding sites]) for these six insect species

Ferré J

Munson P

Van Rie J

Wolfersberger MG

PubMed

ABSTRACT
          
            Cry1Fa insecticidal protein was successfully radiolabeled with
            125
            I-Na. Specific binding to brush border membrane vesicles was shown for the lepidopteran species
            Ostrinia nubilalis
            ,
            Spodoptera frugiperda
            ,
            Spodoptera exigua
            ,
            Helicoverpa armigera
            ,
            Heliothis virescens
            , and
            Plutella xylostella
            . Homologous competition assays were performed to obtain equilibrium binding parameters (
            
              K
              d
            
            [dissociation constant] and
            
              R
              t
            
            [concentration of binding sites]) for these six insect species.
          </jats:p

Carmen Sara Hernández-Rodríguez

Patricia Hernández-Martínez

Jeroen Van Rie

Baltasar Escriche

Juan Ferré

Crossref

Applied and Environmental Microbiology

Specific Binding of Radiolabeled Cry1Fa Insecticidal Protein from Bacillus thuringiensis to Midgut Sites in Lepidopteran Species

Hernández Rodríguez, Carmen Sara

Hernández Martínez, Patricia

Van Rie, Jeroen

Ferré Manzanero, Juan

Repositori d'Objectes Digitals per a l'Ensenyament la Recerca i la Cultura

Specific Binding of Radiolabeled Cry1Fa Insecticidal Protein fromBacillus thuringiensis to Midgut Sites in Lepidopteran SpeciesCarmen Sara Hernández-Rodríguez,a Patricia Hernández-Martínez,a Jeroen Van Rie,b Baltasar Escriche,a and Juan FerréaDepartamento de Genética, Facultad de CC Biológicas, Universidad de Valencia, Burjassot, Valencia, Spain,a and Bayer CropScience, Ghent, BelgiumbCry1Fa insecticidal protein was successfully radiolabeled with 125I-Na. Specific binding to brush border membrane vesicles wasshown for the lepidopteran species Ostrinia nubilalis, Spodoptera frugiperda, Spodoptera exigua, Helicoverpa armigera, He-liothis virescens, and Plutella xylostella. Homologous competition assays were performed to obtain equilibrium binding param-eters (Kd [dissociation constant] and Rt [concentration of binding sites]) for these six insect species.Since the commercial introduction of genetically modifiedcrops for the control of insect pests in 1996, numerous plantspecies of agronomic interest have been transformed to express crygenes from the bacterium Bacillus thuringiensis. Bacillus thurin-giensis Cry1A proteins confer high protection against most of theeconomically important lepidopteran pests, as shown by the un-precedented fast adoption of corn and cotton expressing Cry1Aproteins (Bt corn and Bt cotton, respectively) by growers world-wide (www.isaaa.org). Because some lepidopteran pests are rathertolerant to Cry1A insecticidal proteins, and because of the threatof development of resistance to these proteins, other B. thurin-giensis genes for insecticidal proteins targeting lepidopterans havebeen introduced in plants. Corn expressing Cry1Fa was registeredin 2001 (TC1507 maize; Dow AgroSciences), and cotton express-ing Cry1Fa in combinationwith Cry1Ac has been commercializedsince 2005 (Widestrike; Dow AgroSciences). Cry1Fa has high ac-tivity againstmany lepidopteran species, such asPlutella xylostella,Heliothis virescens, Cacyreus marshalli,Ostrinia nubilalis, Conopo-morpha cramarella, Sesamia nonagrioides, Cydia pomonella,Spodoptera exigua, Spodoptera frugiperda, and Prays oleae (www.glfc.forestry.ca/bacillus).Bacillus thuringiensis Cry proteins exert their toxicity throughbinding to specific sites in the midgut of insect larvae. The affinityfor these sites and their abundance in themidgut contribute to thespecificity and the toxicity of Cry proteins to the target. Further-more, binding competition experiments among Cry proteins areuseful to confirm the physiological basis of cross-resistance and topredict its emergence (1, 2). For example, a common binding sitefor Cry1Fa, Cry1Ja, and Cry1A proteins has been proposed forseveral lepidopteran species based on competition binding assaysamong these proteins, which is in agreement with their cross-resistance patterns (3).Among the different alternatives to label Cry proteins for bind-ing assays, radiolabeling is the only one that allows an accuratecalculation of binding parameters. Furthermore, it provides thehighest sensitivity in competition experiments. Unfortunately, at-tempts at radiolabeling Cry1Fa with 125I-Na were discouraged af-ter the study performed by Luo et al. (5), and labeling of Cry1Fafor binding studies has been since restricted to biotinylation. Luoet al. showed an almost complete reduction in the activity againstS. frugiperda of Cry1Fa and other Cry proteins after labeling with127I (the common and nonradioactive isotope of iodine) accord-ing to a protocol for radiolabeling Cry proteins with the 125I iso-tope with some modifications. The loss of in vivo activity afterlabeling with 127I-Na was accompanied by a lack of specific bind-ing of the 125I-labeled Cry1Fa, which led the authors to concludethat labeling of Cry1Fa with iodine destroyed its biological func-tionality.The aim of the present study was to obtain specific binding of125I-labeled Cry1Fa to brush border membrane vesicles (BBMV)from S. frugiperda larvae and extend it to other lepidopteran spe-cies. We also followed the loss of toxicity of Cry1Fa throughoutthe steps of purification and labeling with the nonradioactive iso-tope under different labeling conditions.The protocol for 125I radiolabeling of Cry proteins indicates amolar ratio of 1:4 of Cry protein to 125I-Na, with a final concen-tration of both Cry protein and NaI on the order of 106 M (7).However, the conditions used by Luo et al. (5) for the labeling ofCry1Fa with 127I were significantly different, with a ratio of Cryprotein to 127I-Na of 1:1,000 (106 M protein and 103 M NaI).The reason for increasing the ratio was probably to minimize thepresence of unlabeled molecules that could contribute to the tox-icity. In our study, the toxicity of trypsin-activated Cry1Fa againstReceived 17 November 2011 Accepted 13 March 2012Published ahead of print 23 March 2012Address correspondence to Juan Ferré, Juan.Ferre@uv.es.Supplemental material for this article may be found at http://aem.asm.org/.Copyright © 2012, American Society for Microbiology. All Rights Reserved.doi:10.1128/AEM.07591-11TABLE 1 Toxicity of trypsin-activated Cry1Fa protein following varioustreatments administered to neonate larvae of S. frugiperda (measuredafter 5 days)aProteinLC50(ng/cm2) 95% FL Slope SEMNonpurified Cry1Fab 51 25–131 0.9 0.1Chromatography-purified Cry1Fab 460c 264–1,040 0.9 0.1127I-Cry1Fa(incubated with 106 M 127I-Na)255c 155–509 1.6 0.1127I-Cry1Fa(incubated with 103 M 127I-Na)1,250a LC50, 50% lethal concentration; FL, fiducial limit at the 95% level.b Protein concentration was estimated by densitometry of Cry1Fa band in SDS-PAGE.c Differences between LC50 values were not statistically significant. The relative potencyof purified Cry1Fa/127I-Cry1Fa was 1.4 (FL of 0.4 to 4.9).4048 aem.asm.org Applied and Environmental Microbiology p. 4048–4050 June 2012 Volume 78 Number 11S. frugiperda was tested after chromatography purification andafter labeling with 127I under two regimens: lowNaI and highNaI.Trypsin-activated and chromatographically purified Cry1Fa (seeFig. S1 in the supplemental material) was labeled with 127I-Na bythe chloramine T method (7) under high or low NaI concentra-tion. Overlay bioassays with artificial diet were performed in du-plicate using seven serial dilutions of Cry1Fa (50 l of each dilu-tion per well). A single neonate larva was placed in each well, witha total of 16 larvae per dilution. Assay plates were incubated at25 2°C, with relative humidity of 65% 5% and a photoperiodof 16:8 (light:dark). Larval mortality was scored after 5 days andanalyzed using the POLO-PC probit analysis program (LeOraSoftware, Berkeley, CA). Toxicity assays showed that the toxicityof Cry1Fa after chromatography purification was reduced ap-proximately nine times with respect to the activity of the nonpu-rified trypsin-activated Cry1Fa (Table 1). The lower toxic activityobtained with the chromatography-purified sample indicates thatthis purification step involves the inactivation of part of Cry1Famolecules. This inactivation may be due to a conformationalFIG 1 Homologous competition binding assays between 125I-Cry1Fa and increasing concentrations of unlabeled Cry1Fa to BBMV from different species. (A)O. nubilalis (0.9mg/ml); (B) S. frugiperda (0.8mg/ml); (C) S. exigua (0.6mg/ml); (D)H. armigera (0.3mg/ml); (E)H. virescens (0.8mg/ml); (F) P. xylostella (0.7mg/ml). Each data point is the mean of two (A, B, and F) or three (C, D, and E) independent replicates. The concentration of BBMV proteins in the reactionmixture is given in parentheses.TABLE 2 Kd (dissociation constant) and Rt (concentration of bindingsites) binding parameters estimated from Cry1Fa homologouscompetition assays with BBMV from O. nubilalis, S. frugiperda,S. exigua, H. armigera, H. virescens, and P. xylostellaInsect speciesMean SEMaKd (nM) Rt (pmol/mg)bO. nubilalis 0.8 0.2 0.44 0.04S. frugiperda 1.5 0.6 0.67 0.11S. exigua 5.3 0.7 1.38 0.14H. armigera 2.0 0.7 0.90 1.16H. virescens 8.3 3.9 1.38 0.30P. xylostella 2.7 1.4 0.27 0.07a Values were obtained from two replicates of the assays with O. nubilalis, S. frugiperda,and P. xylostella and from three replicates of the assays with S. exigua, H. armigera, andH. virescens.b Values are expressed in picomoles per milligram of BBMV protein.Speciﬁc Binding of 125I-Cry1FaJune 2012 Volume 78 Number 11 aem.asm.org 4049change during the processes of binding to and/or release from thecolumn. In addition, important different toxicities of 127I-Cry1Fawere found, depending on the final concentration of 127I-Na usedin the labeling reaction. When a low concentration of NaI wasused (106 M; conditions used for 125I labeling), 127I-Cry1Fashowed no loss of toxicity compared with that of the chromato-graphically purified Cry1Fa (Table 1). In contrast, if the incuba-tion of the toxin was done with a high concentration of NaI (103M; conditions used by Luo et al. [5]), the 127I-Cry1Fa proteinshowed a substantial loss of toxicity (Table 1). Since our resultsindicated that, under common radiolabeling conditions (106 MNaI), Cry1Fa did not lose toxicity against S. frugiperda, Cry1Fawas labeled with 125I-Na and tested for specific binding withBBMV from several lepidopteran species.For binding assays, BBMV were prepared by the differentialmagnesiumprecipitationmethod (8) from last-instar larva guts ofO. nubilalis, S. frugiperda, S. exigua, Helicoverpa armigera, and H.virescens and whole last-instar larvae of P. xylostella. After labelingof Cry1Fa protein with 125I, the estimated specific activity of 125I-Cry1Fa was 0.5Ci/g andwas calculated as previously described(4). Binding parameters, Kd (dissociation constant) and Rt (con-centration of binding sites), were estimated with the LIGANDcomputer program (6). To test for specific binding, increasingamounts of BBMV were incubated with 4.5 nM 125I-Cry1Fa in afinal volume of 0.1 ml of binding buffer (8 mMNa2HPO4, 2 mMKH2PO4, 150 mM NaCl, pH 7.4, 0.1% bovine serum albumin[BSA]) for 1 h at 25°C. An excess (80-fold) of unlabeled toxin wasused to calculate the nonspecific binding. An increase in specificbinding of Cry1Fa was obtained with increasing concentrations ofBBMV from all the species tested (data not shown). Homologouscompetition assays performed with a fixed amount of 125I-Cry1Fa(4.5 nM) and BBMV (see Fig. 1 legend) and increasing concentra-tions of unlabeledCry1Fa confirmed the specific binding of radio-labeled 125I-Cry1Fa (Fig. 1). The competition curves fitted a sin-gle-site model equation and allowed the estimation of Kd and Rtvalues (Table 2). Kd values corresponded to high-affinity bindingsites for Cry1Fa in all the species tested.The successful radiolabeling of Cry1Fa with 125I-Na has di-rectly shown the specific binding of this protein to high-affinitybinding sites in the midgut ofO. nubilalis, S. frugiperda, S. exigua,H. armigera,H. virescens, and P. xylostella. This has allowed us, forthe first time, to estimate the binding affinity and the concentra-tion of binding sites for this protein. Labeling of Cry1Fa with125I-Na will allow the performance of reciprocal heterologouscompetition studies (in which 125I-Cry1Fa is competed by otherunlabeled Cry proteins) to verify receptor models proposed forCry1Fa, such as the occurrence of shared binding sites amongCry1Fa and Cry1A toxins. This information will be very useful forthe sustainable use of B. thuringiensis products combining severalCry toxins as well as for the design of effective transgenic cropsthat would help delay the evolution of insect resistance.REFERENCES1. Ferré J, Van Rie J. 2002. Biochemistry and genetics of insect resistance toBacillus thuringiensis. Annu. Rev. Entomol. 47:501–533.2. Ferré J, Van Rie J, MacIntosh SC. 2008. Insecticidal genetically modifiedcrops and insect resistance management (IRM), p 41–85. In Romeis J,Shelton AM, Kennedy GG (ed), Integration of insect resistant geneticallymodified crops within IPM programs. Springer, New York, NY.3. Hernández CS, Ferré J. 2005. Common receptor for Bacillus thuringiensistoxins Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, Helicoverpazea, and Spodoptera exigua. Appl. Environ. Microbiol. 71:5627–5629.4. Hernández-Rodríguez CS, Van Vliet A, Bautsoens N, Van Rie J, Ferré J.2008. Specific binding of Bacillus thuringiensis Cry2A insecticidal proteinsto a common site in the midgut of Helicoverpa species. Appl. Environ.Microbiol. 74:7654–7659.5. Luo K, Banks D, Adang MJ. 1999. Toxicity, binding, and permeabilityanalyses of four Bacillus thuringiensis Cry1 delta-endotoxins using brushborder membrane vesicles of Spodoptera exigua and Spodoptera frugiperda.Appl. Environ. Microbiol. 65:457–464.6. Munson P, Rodbard D. 1980. LIGAND: a versatile computerized ap-proach for characterization of ligand-binding systems. Anal. Biochem. 107:220–239.7. Van Rie J, Jansens S, Höfte H, Degheele D, Van Mellaert H. 1989.Specificity of Bacillus thuringiensis -endotoxins. Importance of specificreceptors on the brush border membrane of the midgut of target insects.Eur. J. Biochem. 186:239–247.8. Wolfersberger MG, et al. 1987. Preparation and partial characterization ofamino acid transporting brush border membrane vesicles from the larvalmidgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Physiol.86A:301–308.Hernández-Rodríguez et al.4050 aem.asm.org Applied and Environmental Microbiology

Specific binding of radiolabeled Cry1Fa insecticidal protein from Bacillus thuringiensis to midgut sites in lepidopteran species

Specific binding of radiolabeled Cry1Fa insecticidal protein from Bacillus thuringiensis to midgut sites in lepidopteran species

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Repositori d'Objectes Digitals per a l'Ensenyament la Recerca i la Cultura