Figure 1.
Cadherin protein of H. armigera encoded by HaCad.
A. Protein structure of HaCad predicted from cDNA with extracellular region (amino-terminal signal sequence [SIG], 11 cadherin repeats [1]–[11], membrane proximal region [MPR]), transmembrane region (TM), and cytoplasmic domain (C). B. Genomic DNA sequence of HaCad. Resistance allele r1 has a stop codon at 428G in cadherin repeat 3 caused by a genomic DNA deletion of ca. 10 kb [36]. HaCad encoded by resistance allele r15 lacks 55 amino acids in the cytoplasmic domain caused by a 165 bp deletion in exon 32. We found three genomic DNA variants of r15 that cause loss of exon 32, one from each of three field populations: 1459 bp insertion from Xiajin, 92 bp deletion from Anyang, and >5000 bp insertion from Anci.
Figure 2.
Responses to Bt toxin Cry1Ac of H. armigera from a susceptible strain (SCD, blue), three resistant strains (red), and the F1 progeny from crosses between each resistant strain and the susceptible strain (purple).
SCD-r1: resistant strain with allele r1 affecting the extracellular domain of HaCad. XJ-r15 and AY-r15: resistant strains (from Xiajin and Anyang, respectively) with allele r15 affecting the cytoplasmic domain of HaCad. Resistance ratio is the concentration killing 50% of larvae (LC50) of each strain or group of F1 progeny divided by the LC50 for the susceptible SCD strain. The black bars show the 95% fiducial limits for LC50.
Table 1.
Magnitude and dominance of resistance to Cry1Ac in H. armigera associated with cadherin resistance alleles: r15 in the cytoplasmic domain and r1 in the extracellular region.
Figure 3.
Genetic linkage between the cytoplasmic domain mutant of HaCad (r15) and resistance to Cry1Ac in the XJ-r15 strain of H. armigera.
We crossed a female (ss) from the susceptible SCD strain with a male from the resistant XJ-r15 strain (r15r15) to produce the F1 family (r15s). Next we crossed an F1 male (r15s) with a susceptible SCD female (ss) to produce a backcross family from which larvae were placed on untreated diet (control) or diet treated with either 0.3 or 0.5 µg Cry1Ac per cm2. After 5 days, all survivors were transferred to untreated diet, reared to the final instar, and genotyped. The frequency of heterozygotes (r15s) relative to susceptible homozygotes (ss) was significantly higher for survivors on treated diet (68∶5) than for survivors on untreated diet (27∶23) (Fisher's exact test, P<0.0001).
Figure 4.
Cry1Ac binding to Sf9 cells transfected with four alleles of HaCad.
s: susceptible allele. r15: resistant allele, encoding cadherin with a 55 amino acid deletion in the cytoplasmic domain (C). s/r15: chimeric allele with C from r15 and the other components from s. r15/s: complementary chimeric allele with C from s and the other components from r15. Cells were treated with 10 nM Cry1Ac, then probed sequentially with anti-Cry1Ac antiserum (1∶100) and FITC-conjugated anti-rabbit antibody (1∶100). No Cry1Ac binding was detected in control cells that were either transfected with an empty bacmid (EB) or not transfected (Sf9).
Figure 5.
Mortality of Sf9 cells exposed to Cry1Ac.
Sf9 cells were transfected with one of four alleles of HaCad (s, r15, r15/s, and s/r15; see Figures 4 and S2 for details) or an empty bacmid (EB), or were not transfected (NT). For cells transfected with alleles of HaCad, LC50 values (95% FL) were significantly higher for alleles with the cytoplasmic domain of r15 (r15: 85 [71–110] and s/r15: 82 [68–100]) than for alleles with the cytoplasmic domain of s (s: 38 [31–46] or r15/s: 38 [31–45]). LC50 values did not differ significantly between Sf9 cells transfected with alleles of HaCad that had the same cytoplasmic domain (r15 and s/r15; s and r15/s).