Hemocyte response to treatment of susceptible and resistant Asian corn borer (Ostrinia furnacalis) larvae with Cry1F toxin from Bacillus thuringiensis

Abstract

Midgut receptors have been recognized as the major mechanism of resistance to Cry proteins in lepidopteran larvae, while there is a dearth of data on the role of hemocyte's response to Cry intoxication and resistance development. We aimed at investigating the role of circulating hemocytes in the intoxication of Cry1F toxin in larvae from susceptible (ACB-BtS) and resistant (ACB-FR) strains of the Asian corn borer (ACB), Ostrinia furnacalis. Transcriptome and proteome profiling identified genes and proteins involved in immune-related (tetraspanin and C-type lectins) and detoxification pathways as significantly up-regulated in the hemocytes of Cry1F treated ACB-FR. High-throughput in vitro assays revealed the binding affinity of Cry1F with the tetraspanin and C-type lectin family proteins. We found significant activation of MAPKinase (ERK 1/2, p38α, and JNK 1/2) in the hemocytes of Cry1F treated ACB-FR. In testing plausible crosstalk between a tetraspanin (CD63) and downstream MAPK signaling, we knocked down CD63 expression by RNAi and detected an alteration in JNK 1/2 level but a significant increase in susceptibility of ACB-FR larvae to Cry1F toxin. Information from this study advances a change in knowledge on the cellular immune response to Cry intoxication and its potential role in resistance in a lepidopteran pest.

Keywords: C-type lecin; CD63; Cry1F; Ostrinia funacalis; hemocyte immune function.

Figure 1

Differential gene expression after Cry1F treatment in hemocytes from susceptible (ACB-BtS) and Cry1F-resistant (ACB-FR) strains of Asian corn borer. (A) Hemocyte sample processing and enriched genes grouped after KEGG analysis. The numbers shown on the right side of every horizontal bar represent the number of genes involved in that specific pathway. Asterisks represent significant enrichment of genes in the hemocytes of Cry1F- treated ACB-BtS and ACB-FR larvae compared with respective non-treated controls (Student’s t-test, P<0.05). (B) Gene Ontology analysis shows the number of significantly enriched genes in the hemocytes of Cry1F treated ACB-BtS and ACB-FR larvae compared to respective non-treated controls (Student’s t-test, P<0.05).

Figure 2

Analysis of differentially expressed genes encoding membrane and immune/detoxification proteins in hemocytes after Cry1F treatment in susceptible (ACB-BtS) and Cry1F-resistant (ACB-FR) strains of Asian corn borer. (A) Differentially expressed genes (DEGs) and fold change compared with respective controls (Student’s t-test, P<0.05). The numbers in the heat map represent the log2 fold change in gene expression, a value of 0 indicates that the gene was not detected in the DEGs or down-regulated lists. (B) Difference (fold change) in expression levels (mean ± standard error) of genes involved in detoxification and immune pathways of hemocytes from the ACB-BtS and ACB-FR strains after Cry1F treatment (LC50 dose). Asterisks denote that differences in gene expression were statistically significant (Student’s t-test, **P<0.01, NS, not significant).

Figure 3

Glutathione S-transferase (GST) activity in hemolymph of susceptible and resistant Asian corn borer (ACB) larvae after exposure to Cry1F (LC₅₀ dosage: ACB-BtS - 1.2 μg/g and ACB-FR 1,075 μg/g, toxin/diet). Asterisk represents statistically significant difference (Student’s t-test, *P<0.05, **P<0.01), NS, not significant.

Figure 4

Differential protein levels detected in hemolymph from ACB-BtS and ACB-FR larvae after treatment with Cry1F. (A) Hemolymph sample processing and Volcano plot showing the number of proteins with significantly increased (red dots) or reduced (green dots) abundance in the hemolymph of Cry1F-treated ACB-BtS and ACB-FR larvae compared to controls (Student’s t-test, P<0.05). (B) Differentially abundant proteins (DAPs) in hemolymph from Cry1F treated ACB-BtS and ACB-FR larvae involved in developmental functions and immune pathways. Asterisks in the ACB-BtS column indicate proteins with no significant change in abundance (Student’s t-test, P<0.05). A value of 0 indicates the protein was not detected in the DAPs list. (C) Enriched (gene ontology) hemolymph proteins according to biological process and molecular functions in hemocytes from ACB-BtS and ACB-FR larvae after Cry1F treatment. Pathways and respective functions marked in a blue box indicate enrichment of proteins with immune function (-Log10, P-value).

Figure 5

Domain-based protein-protein interaction (PPI) in silico analysis of C-type lectins and tetraspanin with domain II of Cry1F. (A) Protein domains identified in C-type lectins, tetraspanin and Cry1F by NCBI Conserved domain, Pfam or InterProScan. (B) The PPI complexes of Cry1F with C-type lectins and tetraspanin (β = Central β-sheet domain II of Cry1F, CBD 1 & D2 = Carbohydrate-binding domains, EC 2 - ectodomain EC 2).

Figure 6

Pull-down assays testing protein-protein interactions (PPI) between Cry1F, C-type lectins and tetraspanin. Each panel presents SDS-PAGE gels initially stained with FastBlue Protein stain (left) and further stained using ProteoSilver silver staining kit (right). Lanes M - TrueColor protein marker, Lanes 1 - Bait flowthrough, Lanes 2 - Prey flow through, Lanes 3 - Final elution, Lanes 4 - Purified bait protein, Lanes 5 - Prey protein (activated Cry1F) and Lanes 6 - Non-treated control. The arrow mark indicates detection of Cry1F protein in the elution, supporting interaction with immobilized bait proteins. ACB proteins tested: Macrophage mannose receptor 1-like (MMR1), C-type mannose receptor 2-like (C-MR2), CD209 antigen-like protein E (CD209), C-type lectin lectoxin-Phi1-like (C-lectoxin), hemolymph lipopolysaccharide-binding protein (LBP), and CD63 antigen-like (CD63 - ectodomain EC2).

Figure 7

Activity of selected mitogen-activated protein kinases (MAPKs) in hemocytes from ACB-BtS and ACB-FR larvae in response to Cry1F treatment. MAPK activity (mean and corresponding standard errors from three biological replicates) in hemocyte lysates after 48 h of treatment with Cry1F toxin (1,075 μg/g; toxin/diet). Antibody cocktails detected MAPK family kinase enzymes ERK 1/2, p38α, and JNK 1/2. Different letters on vertical bars represent statistically significant differences between treatments for the specific gene tested (One Way ANOVA and post hoc LSD Tukey test; P < 0.05).

Figure 8

Functional testing of CD63 tetraspanin as putative Cry1F toxin receptor in ACB-FR using silencing by RNAi. (A) Amplified PCR product of CD63 gene after RNAi at different time points and compared with internal controls. (B) Relative mean fold CD63 expression ( ± SE) from three biological replicates detected using qRT-PCR. Different letters represent significantly different transcript levels (Tukey test, P< 0.05). (C) Mean percentage mortality after 48 h in ACB-FR larvae exposed to control or diet with Cry1F alone or in the presence of dsRNA targeting GFP or CD63. Shown are means and corresponding SE estimated from six independent bioassays represented by red circles. Different letters represent significant differences (Tukey test, P< 0.05). (D) MAPK activity in hemocyte lysates after CD63 gene knockdown and treatment with Cry1F (LC₅₀ dose) for 48 h in the ABC-FR strain, compared with control group. Blank values were subtracted from the respective experiments. Vertical bars under different letters were statistically significant and same letters were not significant (One Way ANOVA and post hoc LSD Tukey test, P<0.05).