Roles of Midgut Cadherin from Two Moths in Different Bacillus thuringiensis Action Mechanisms: Correlation among Toxin Binding, Cellular Toxicity, and Synergism

Abstract

The midgut cadherin has been described as one of the main functional receptors for Bacillus thuringiensis (Bt) toxins. Plutella xylostella (P. xylostella) and Helicoverpa armigera (H. armigera) are two major target pests of Bt toxins in China, and the roles of their cadherins in the action of Bt toxins have been only partially studied. Here, we expressed the two cadherins in Sf9 cells and their partial extracellular domains in Escherichia coli and tested them for Bt toxin binding, cellular toxicity, and synergism with toxins. Our results suggested that PxCad might function as a Cry1Ac receptor, although it showed lower binding levels to Cry1Ac and reduced cytotoxicity compared with HaCad. PxCad and HaCad are not receptors for Cry2A, Cry1B, Cry1C, and Cry1F toxins, although some of them can bind to the cadherins. The PxCad-TBR exhibits higher enhancement of Cry1Ac and weak enhancement of Cry1F toxicity in P. xylostella larvae, although it is not the receptor of Cry1F.

Evidence of a shared binding site for Bacillus thuringiensisCry1Ac and Cry2Aa toxins in Cnaphalocrocis medinalis cadherin

“…The CmCad protein showed typical topology for Cry receptor cadherins in Lepidoptera (Chen et al, 2009; Fabrick et al, 2009;Flannagan et al, 2005;Park and Kim, 2013), consisting of 11 extracellular CRs and transmembrane and CYTs. Sequential analysis of CmCad-derived peptides localized the Cry TBR to the CmCad-CR9-11 (T1211-L1456) region, which is homologous to the Cry1Ac TBR previously described in cadherins from H. armigera and P. xylostella (Gao et al, 2019). Interestingly, the CmCad-CR9-11 peptide displayed highaffinity binding by both Cry1Ac and Cry2Aa, suggesting that a shared binding site exists in this region.…”

“…Lyophilized activated Cry1Ac, Cry1B, Cry1C, Cry1F and Cry2Aa toxins were purchased from Envirotest-China (an agent of Envirologix Inc., Portland, ME, USA), and solubilized in sodium carbonate buffer (pH 9.6), aliquoted and stored at À80 C until used. Soluble Cry toxins were biotinylated with the EZ-Link Sulfo-NHS-SS-Biotinylation kit (Thermo Fisher Scientific) as described before (Gao et al, 2019). Briefly, 28.4 μl of 10 mM biotin was used to label 1 mg Cry toxin in a 1 ml final volume reaction.…”

“…In lepidopteran cadherins, Cry toxin binding regions (TBRs) map primarily within the six CR domains proximal to the membrane (such as CR6-11/CR7-12) plus the MPED (Fabrick and Wu, 2015). For instance, we identified that a fragment of Plutella xylostella cadherin (T1202-I1447, PxCad-TBR) and its homologous fragment from Helicoverpa armigera (T1217-L1461, HaCad-TBR) bound the Cry1Ac toxin (Gao et al, 2019). Heterologously expressed cadherin fragments containing TBRs bound Cry toxins with high affinity, promoted oligomerization and enhanced Cry toxicity in some lepidopterans (Chen et al, 2007;Fabrick et al, 2009;Pacheco et al, 2009b;Park et al, 2009Park et al, , 2019.…”

Neuropeptide F from endocrine cells in Plutella xylostella midgut modulates feeding and synergizes Cry1Ac action

“…A susceptible strain of P. xylostella (HN) was purchased from Keyun and has been maintained in the laboratory without exposure to insecticides or Bt toxins for more than 5 years (Gao et al, 2019). Larvae of P. xylostella were reared on an artificial diet based on wheat germ powder at 27 ± 1°C with a 14:10 (L:D) photoperiod.…”

The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda

“…For instance, it has been reported that the cadherin from Plutella xylostella (PxCad) is not associated with resistance in P. xylostella to Cry1Ac [17]. However, other reports suggest that PxCad is a functional receptor of Cry1Ac, since PxCad can increase cytotoxicity of Cry1Ac when expressed in the Sf9 cell line [18][19][20]. In addition, we reported that Spodoptera litura cadherin (SlCad), in contrast to HaCad, cannot increase cytotoxicity of Cry1Ac when expressed in Hi5 cells, suggesting that SlCad is not a functional receptor of Cry1Ac in S. litura [14].…”