Vol.12_No.2 - Pesticide Alternatives Lab - Michigan State University

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Mba, R.E.C., Stephenson, P., Edwards, K., Melzer, S., Mkumbira, J.,Gullberg, U., Apel, K., Gale, M., Tohme, J. and Fregene, M. 2001.Simple Sequence Repeat (SSR) Markers Survey of the Cassava(Manihot esculenta Crantz) Genome: Towards an SSR-BasedMolecular Genetic Map of Cassava. Theoretical and AppliedGenetics Journal. 102:21-31.A. Bellotti, B. Arias, A. Bohorquez, J. Vargas,G. Trujillo, C. MBA, M. C. Duque, & J. TohmeCentro Internacional de Agricultura Tropical (CIAT)AA6713, CaliColombiaandH. L. VargasCORPOICAEspinal, TolimaColombiaEvidence for Multiple Mechanisms of Resistance to Cry1Ac and Cry2A Toxins from Bacillus thuringiensis inHeliothis virescensBacillus thuringiensis (Bt) is a common sporeformingbacterium that produces insecticidal proteinscalled Cry toxins (from Crystal). Thecommercialization of transgenic plants producing Crytoxins has greatly affected insect control methods dueto their environmental safety and increased crop yield.In 1996, transgenic cotton plants producing Cry1Actoxin were commercialized to control Heliothisvirescens (tobacco budworm). This insect is one of themost important pests of cotton, among other crops. Aswith any insect control method, development ofresistance to Bt toxins is one of the main concerns onthe wide use of transgenic Bt plants. Although no H.virescens resistance episodes to Bt cotton have beenreported in the field so far, laboratory resistanceselection of H. virescens has demonstrated that thegenetic potential for resistance development exists(Gould et al., 1992, 1995). The study of resistance inthese laboratory-selected insect strains has helped toidentify potential resistance mechanisms and strategiesaimed to manage and delay the onset of resistance.Disruption of any step in the mode of action of Bttoxins can result in resistance to these toxins. Thegeneral mode of action of Bt toxins includes ingestionby the susceptible insect, solubilization and activationto toxic forms by insect midgut enzymes, binding andinsertion into the membrane of the midgut epithelium,and midgut cell lysis by osmotic shock (Knowles,1994). Although several mechanisms of resistance toBt toxins in laboratory-selected insects have beenproposed, alteration of toxin binding to midgutreceptors is the best studied (Ferré and Van Rie, 2002).Since an insect is less likely to develop resistanceto two toxins with distinct modes of action, one of theproposed methods to delay the onset of resistance to Btplants in the field is the generation of transgenic linesexpressing different Bt toxins in combination (Gould,1998). To assure the efficacy of this approach thetoxins selected for expression should not sharecommon binding sites and must have distinct modes ofaction.In brush border epithelium membrane vesicles(BBMV) from H. virescens, Cry1Aa, Cry1Ab, Cry1Ac,Cry1Fa, and Cry1Ja toxins share a common bindingsite (receptor A). Cry1Ab and Cry1Ac have anadditional binding site (receptor B) and Cry1Ac is theonly toxin that can recognize a third binding site(receptor C) (Van Rie et al., 1989; Jurat-Fuentes andAdang, 2001). According to this model of bindingsites, alteration of receptor A would potentially lead toreduced binding and possibly resistance to all Cry1A,Cry1Fa and Cry1Ja toxins. This mechanism wasproposed to occur in the Cry1Ac-selected YHD2 strainof H. virescens (Lee et al., 1995).One of the most important toxin candidates to beused in combination with Cry1Ac in Bt cotton tocontrol H. virescens is Cry2A. This toxin does notshare binding sites with Cry1A toxins (Jurat-Fuentesand Adang, 2001) and has a distinct mode of action(English et al., 1994; Morse et al., 2001). TransgenicBt cotton plants expressing both Cry1Ac and Cry2Ahave been shown to enhance control of H. virescens(Stewart et al., 2001). Interestingly, the Cry1Aclaboratory selected CP73-3 and KCB H. virescensstrains developed cross-resistance to Cry2A, amongother toxins (Gould et al., 1992; Forcada et al., 1999).These strains were backcrossed to susceptible insectsand the offspring were selected with Cry2A to increaseresistance to this toxin. This selection regime led to thegeneration of the CXC (derived from CP73-3) andKCBhyb (derived from KCB) strains, which showedincreased Cry2A and Cry1Ac resistance levels whencompared to their parental strains (Kota et al., 1999).Both strains were also cross-resistant to Cry1Aa,Cry1Ab, and Cry1Fa toxins (F. Gould, unpublishedobservation).To study the mechanism of resistance in the CXCand KCBhyb strains, we performed toxin-bindingassays with radio labeled Cry1A toxins. BBMV fromYDK (susceptible control strain), CXC and KCBHybinsects were isolated and incubated with increasingconcentrations of labeled Cry1A toxins to generate abinding saturation curve for each Cry1A toxin.42