Viral and bacterial strains and insect culture
In the study, 2 viral and a bacterial chitinase proteins were investigated in terms of their potential activity. Autographa californica nucleopolyhedrovirus (AcNPV) and Cydia pomonella granulovirus (CpGV) species were used for viral chitinase sources. Bacterial chitinase protein (chitinase C) belongs to Serratia marcescens that was previously isolated from dead Helicoverpa armigera (Hubner, 1808) larvae. The S. marcescens chitinase gene (accession number: KF823632) was provided cloned into the pET-28a (+) expression vector (Danişmazoğlu et al. 2015). Lab-reared culture of Galleria mellonella (Linnaeus, 1758). was used in the activity assay. G. mellonella larvae were maintained on an artificial diet (22% glycerol, 24% wheat bran, 22% honey, 24% honeycomb, and 4% water) and reared at 26 ± 2°C, and 60–70% relative humidity, with a 14:10-h light:dark photoperiod.
In silico analysis of chitinases
AcNPV (NC_001623.1), CpGV (NC_002816.1), and S. marcescens (KF823632) chitinase proteins were analyzed in terms of their domain content at the NCBI conserved domain search database. Amino acid sequences of chitinases were compared to each other using NCBI protein blast and multiple alignment tools.
Construction of chitinase expressing bacterial vectors
Viral genomic DNAs were isolated from virus suspensions, using DNeasy Blood & Tissue Kit (Qiagen, 69506). Specific primers were designed for both AcNPV chitinase (Fw: 5′-GGGGATCCATTCCCGGCACGC-3′; Rv: 5′-GGCTCGAGTTACAGTTCATCTTTAGGT-3′) and CpGV chitinase (Fw: 5′-GGGGATCCAAACCCGGCACACC-3′; Rv: 5′-GGCTCGAGTCATACTGAATTGCACAC-3′). BamHI and XhoI restriction endonucleases were added to the 5′ ends of forward and reverse primers, respectively. Both chitinase genes (AcNPV-chi, CpGV-chi) were amplified by PCR from genomic DNAs. The reactions contain 10 ng of genomic DNA, 2.5 μl of both primers of 10 μM, 1 μl of 10 mM dNTPs, 10 μl 5X Phusion HF reaction buffer, and 1 U Phusion HF DNA polymerase. Lastly, the volume was adjusted to 50 μl with sterile dH2O. The reaction program was as follows: an initial denaturation step at 98°C for 3 min followed by 30 cycles of 98°C for 50 s, 55°C for 50 s, 72°C for 1 min, and final extension step at 72°C for 10 min. The PCR products were ligated into the pJET1.2/blunt vector according to the manufacturer’s instructions. Ligation reactions were transformed into E. coli DH10β cells, and positive clones were confirmed. The generated recombinant plasmids carrying AcNPV-chi and CpGV-chi genes were named as pAcNPV and pCpGV, respectively. The chitinase genes were sequenced by Macrogen Inc. (Amsterdam, the Netherlands). Following the sequence analysis, both chitinase genes containing BamHI and XhoI restriction sites at their 5′ and 3′ ends were introduced into the pET-28a(+) expression vector using the same sites. These plasmids were transformed into E. coli BL21 (DE3) competent cells, and recombinant plasmids were selected in the presence of Kanamycin (50 μg/ml).
Over-expression, purification, and western blot analysis of the recombinant proteins
The AcNPV-Chi, CpGV-Chi, and S. marcescens-ChiC proteins were overexpressed as fusion proteins with the 6×His-tag at their N-terminal in E. coli BL21 (DE3) cells. Chitinase proteins were purified by using the MagneHis™ Protein Purification System Kit (Promega) and dialyzed for 24 h through 1 l of 1X PBS buffer, pH 7.5. The identification and purity of the samples were confirmed by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and subsequently Coomassie staining. Western blot analysis was also performed to demonstrate heterologous gene expression at the immunological level. Electrophoresed proteins were transferred to the nitrocellulose membrane. Immune-detection was performed using 1:1000 diluted polyclonal rabbit anti-His-taq antibodies (Abcam) and subsequently 1:1000 diluted polyclonal alkaline phosphatase-conjugated goat-anti-rabbit IgG (Millipore). The binding of the antibodies was then visualized with the NBT-BCIP substrate system (Roche).
Chitinase activity assay
Chitinase activities were assayed by using the colorimetric 3,5-dinitrosalicylic acid (DNS) method (Monreal and Reese 1969) with some modifications. The purified bacterial and viral chitinases (0.5 μg) were reacted with 150 μl of colloidal chitin (12.5 mg/ml chitin) as a substrate and incubated at 30°C for 3 h. The reactions were terminated by boiling the mixture in the presence of 300 μl DNS acid reagent for 5 min. In the control reaction, only substrate and DNS were used. The hydrolysis of chitin was measured at 540 nm and run with a glucose standard. One unit of the chitinase activity was defined as the amount of enzyme that liberates 1 μM of reducing sugar per 1 min at 30°C.
Potential activity test
Proteins were tested on G. mellonella at 5 different concentrations (1.5, 1.8, 2, 2.5, and 3 μg protein) per larvae. Bioassays were performed by 30 larvae (3rd instar) for each concentration in triplicate. The larvae were starved for 12 h and then fed with an artificial diet inoculated with desired concentrations of protein. After consumption of the inoculated diets, non-inoculated ones were added and incubated at 26 ± 2°C. Control group larvae were fed on only 1X PBS treated diets. Mortalities were recorded daily for 14 days. An experimental design, showing the insecticidal activity tests, is given in Supplementary Fig. 1.
Statistical analysis
Mortality data were corrected using Abbott’s formula (Abbott 1925); LC50 and LT50 values were calculated by Probit analysis using MS Excel (Finney 1952).
