Figure legends
Figure 1 . The workflow of on-membrane capture
Figure 2 . DBM midgut proteins revealed as binding candidates of Bt toxins . (A) Peptide identification analysis on a Q-Exactive Orbitrap LC-MS system (60 min gradient). (B) Distribution of molecular weight and isoelectric point of DBM midgut proteins. (C) Venn diagram showing DBM midgut proteins captured by three Bt toxins Cry1Ac, Cry1Ab, and Cry1Bd.
Figure 3 . DBM GSSs bind directly to Cry1Bd . (A) Binding of GSS1 and GSS2 to Cry1Bd was detected by pull-down assay. His-GSS1 or His-GSS2 attached to the cobalt resin was incubated with one of Bt toxins Cry1Ac, Cry1Ab, and Cry1Bd. Cry1Ac or Cry1Ab was not eluted with either GSSs, and Cry1Bd was co-eluted with both GSS1 and GSS2. (B) Binding of GSS1 and GSS2 to Cry1Bd was detected by far-western blot. Bt proteins on nitrocellulose membranes were denatured and renatured by gradually reducing the guanidine-HCl concentration, then incubated with 5 µg His-GSS1 or His-GSS2 after the membrane was blocked. His-tagged GSSs was detected by anti-His antibody with Cry1Bd and not with Cry1A or Cry1Ab.
Figure 4. Reduction in GSSs expression increases tolerance of DBM Bt toxins . (A and B) Expression of GSS1 andGSS2 in DBM were knocked down. Freshly hatched DBM larvae were fed leaves of either dsGFP or dsGSS1 lines for about 7 d until larvae reached the third instar, whereupon they were harvested for RNA analysis. Values (mean ± SD) were obtained from three independent experiments. ** above the columns indicates statistical significance between samples (P < 0.01). (C, D, E) LC50 of GSS -silenced larvae to three Bt toxins Cry1Ac, Cry1Ab, and Cry1Bd. Two leaves taken from 4-week-old A. thaliana plants of genotypes dsGFP or dsGSS1 were laid on a moistened filter paper in a 15 mm petri dish. Freshly hatched DBM larvae were placed on leaves of each genotype and fed for 7 d. On the eighth day, larvae were transferred to fresh leaves coated with a diluted suspension of Bt proteins in HEPES buffer (pH 8.0), or HEPES buffer as a control. Mortality was recorded after 24 h and LC50 was calculated by probit analysis based on the dose determined to be high enough to kill 100% of larvae. Values (mean ± SD) were obtained from three independent experiments. ** above the columns indicates statistical significance between samples (p < 0.01).
Figure 5. DBM GSSs are susceptible factors to Cry1Bd . (A ) DsRed fluorescence phenotypes of hybrid offspring produced by transformed silkworms and wild type Nistari. Insects with DsRed fluorescence (indicated by arrow) were selected for Cry1Bd toxicity tests. (B ) LC50 of hybrid silkworm against Cry1Bd. Four squares of mulberry leaf (4 × 4 cm), coated with 50 μL of a diluted suspension of Cry1Ab protoxin in HEPES buffer (pH 8.0), were fed to 10 second instar larvae for 24 h. The dose high enough to kill 100% of susceptible larvae was determined. Probit analysis was carried out using SPSS to determine the LC50 value. Values (mean ± SD) were obtained from three independent experiments. ** indicates statistical significance between samples (p < 0.01).
Supplemental Table 1 List of primers used in this study.
Supplemental Table 2 List of proteins captured by Cry1Ac, Cy1Ab, and Cry1Bd via the on-membrane capture.
Supplemental Figure 1 The peptide244RIFAAMVK252, identified by MS in the Cry1Bd sample, matched GSS1 and GSS2.
Supplemental Figure 2 GPI-anchor sites of GSS1 (A) and GSS2 (B) predicted by GPI Modification Site Prediction.
Supplemental Figure 3 Transmembrane helices of GSS1 (top) and GSS2 (bottom) analyzed by TMHMM. Both GSS1 and GSS2 have N-terminal secretory signal peptides, indicating that they are extracellular proteins. GSS2 also contains a transmembrane helix at its N-terminal, indicating that it is a membrane anchor protein with a C-terminal extending outside of the cell membrane.
Supplemental Figure 4 Genomic insertion of DBM GSS1 (A) and DBM GSS2 (B) in transgenic silkworm. Genomic insertion ofGSS1 and GSS2 in transgenic silkworm lines, as revealed by inverse PCR and sequencing. The transgene integration site inGSS1 lies in chromosome 23, between two genes KAIKOGA026485 and KAIKOGA026486. The transgene integration site in GSS2 lies in chromosome 11, between BMgn01916 and BMgn011917. Chromosome localization and partial genomic DNA sequences between the Sau3AI site and the 3ʹ or the 5ʹ insert boundaries of the vector are shown. In all insertions, the TTAA insertion site found in canonical piggyBac insertions was found at the 3ʹ and 5ʹ insert boundary.