Cry64Ba and Cry64Ca, Two ETX/MTX2-Type Bacillus thuringiensis Insecticidal Proteins Active against Hemipteran Pests

Strains and plasmids.Bt strain 1012 (IPPBIOTSUC1012, China General Microbiological Culture Collection Center [CGMCC] strain 10783) was isolated from a soil sample from Baiwangshan Forest Park in Beijing and preserved at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (IPPCAAS). Scanning electron microscopy of the spore-crystal mixture of Bt strain 1012 was performed following the process described by Shu et al. (26). The spore-crystal mixtures were washed in sterile distilled water and then resuspended in 5 volumes of solubilization buffer (50 mmol/liter Na₂CO₃-NaHCO₃ and 50 mmol/liter EDTA; pH 10.0) for 1 h at 37°C. Then 2× Laemmli sample buffer was added, and the samples were boiled for 5 min, centrifuged at 12,000 × g for 5 min, and loaded onto a 12% SDS-polyacrylamide gel. Escherichia coli DH5α was used for common transformation. E. coli SCS110 was used to produce the nonmethylated recombinant plasmids that were used for Bt transformation. HD73⁻, a Bt acrystalliferous strain, was used as the host strain for protein expression. The vector pHT-1A, which contains the pHT315 sequence, was used as the expression vector (27) linked with the promoter of cry1Ac. The attP site and ccdB gene were integrated into the plasmid for Gateway reaction and positive clone selection. All E. coli strains were cultured at 37°C in Luria-Bertani (LB) medium. Bt strains were incubated at 30°C with shaking in 1/2 LB medium (0.5% NaCl, 0.5% tryptone, and 0.25% yeast extract). Erythromycin (Erm) and ampicillin (Amp) were used for bacterial selection at concentrations of 50 and 100 μg/ml, respectively. Erm was a selection marker for Bt strains, and Amp was a selection marker for E. coli strains.

Preparation of genomic DNA, DNA sequence, and computational analysis.Genomic DNA was extracted from Bt 1012 following the method described by Song et al. (28). The genome sequencing was performed on an Illumina HiSeq 2500 platform. The short sequence reads were assembled with SOAPdenovo (29). The open reading frames (ORFs) were predicted using GeneMark (30). A local Cry toxin database was established by available quaternary rank model amino acid sequences in the list of delta-endotoxins on the Bt toxin nomenclature website (15) and used for the Bt 1012 genome toxin gene annotation by the BLASTP program with a cutoff E-value of 10⁻⁶. The phylogenetic tree was constructed by using the distance-based neighbor-joining method with MEGA 7.0 (31). SWISS-MODEL workspace was used to model the three-dimensional structure of Cry64Ba (32). Multiple alignments were performed by using Clustal W analysis (33). Protein structure comparison was performed by using the TM-align algorithm (34). The hydrophilic/hydrophobic amino acids were indicated by the Kyte-Doolittle method (35).

Transcriptional analysis.The promoter and terminator predictions were performed using BPROM (http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) and FoldRNA (http://www.softberry.com/berry.phtml?topic=foldrna&group=programs&subgroup=rnastruct) on SoftBerry online programs. Total RNA was extracted from Bt 1012 after 48 h cultivation on LB agar plates using TRIzol following the manufacturer's instructions. DNase digestion was carried out to avoid contamination with genomic DNA, followed by the phenol-chloroform method to isolate the total RNA. cDNA was synthesized using PrimeScript RTase (TaKaRa, China) according to the manufacturer's instruction. Primers used to detect the transcription units are shown in Table 2. The thermal program of PCR was as follows: incubation at 95°C for 2 min and then 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 2 min with a final extension at 72°C for 10 min.

Gene cloning of Bt toxins from strain 1012.The cry64 genes were amplified from Bt 1012 genomic DNA using specific primers (Table 2) with an attB1 site (5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CT-3′) and an attB2 site (5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3′) added to the 5′ end of the forward and reverse primers, respectively. The fragment containing both cry64Ba and cry64Ca was amplified with the Cry64BaF and Cry64CaR primers (Table 2). Gateway BP recombination reactions between cry gene fragments and pHT-1A were performed using BP Clonase enzyme mix (Thermo Fisher, USA) following the manufacturer's instruction. The recombinant products were transformed into E. coli DH5α. Positive clones were selected and verified by a 3730xl DNA sequencer (Applied Biosystems, USA).

Protein expression and purification.The recombinant plasmids were demethylated by transformation into E. coli strain SCS110. Purified plasmid from this strain was electroporated into Bt acrystalliferous strain HD73⁻ for protein expression. Positive clones were selected by PCR. The method for protein extraction from recombinant strains was described by Zhou et al. (36). The recombinant strains were grown in 1/2 LB until 50% of the crystal was released. The spores and crystals were collected by centrifugation at 8,000 × g for 10 min at 4°C. After being washed with 1.0 mol/liter NaCl and distilled water, the mixture was resuspended in solubilization buffer incubated on ice (shaking at 100 rpm) for 1 h. The supernatant was collected by centrifugation at 12,000 × g for 10 min and then adjusted to pH 5.0 using 4.0 mol/liter sodium acetate-hydrogen acetate and kept at 4°C for 1 h. The pellet was harvested by centrifugation at 13,000 × g for 15 min, washed with distilled water twice, and dissolved in 50 mmol/liter Na₂CO₃ (pH 10). To purify the Cry64Ba/Cry64Ca proteins, an ion-exchange chromatography was conducted with HiTrap Q HP (GE Healthcare, USA). Target proteins were eluted with a linear ion strength gradient of 0 to 0.5 mol/liter NaCl included in the protein buffer, followed by gel filtration chromatography with HiPrep 26/60 Sephacryl S-100 HR columns (GE Healthcare). BSA was used as a molecular weight standard in gel filtration chromatography. Tris-HCl (20 mmol/liter; pH 8.0) was used as the mobile phase, with a flow rate of 0.5 ml/min. The protein concentration of the purified Cry64Ba/Cry64Ca complex was determined by ImageJ using BSA as a standard.

Bioassays.Liquid artificial diet for planthopper feeding was prepared according to Fu et al. (37). Based on the bioassay method described by Wang et al. (38), the container used in the experiment was improved in two points. First, we used a polyethylene terephthalate (PET) cylindrical bottle 7 cm in height and 4 cm in diameter with a screw cap. Two holes were opened in the bottle. The hole on the sidewall had a diameter of 6 mm and was covered with micropore tape pasted inside the bottle for ventilation; the other hole was on the cap to support the membrane-feeding package and had a diameter of 3 cm. Second, 1 ml 2% agarose gel was put on the bottom of the bottle to maintain a suitable humidity for rearing L. striatellus. Twenty L. striatellus nymphs (3rd instar) were put into the bottle. The liquid diet was replaced every 2 days. Mortality was recorded after 6 days of bioassay. The bioassay method for S. furcifera was same as above.

One-dose bioassay tests of the Cry64Ba/Cry64Ca protein (300 μg/g) against O. furnacalis (39), P. xylostella (40), and C. bowringi (41) were conducted as described. The newly hatched O. furnacalis moths were exposed to the semiartificial diet containing Cry64Ba/Cry64Ca toxin that was dispensed into 48-well trays. One neonate was added per well with 0.15 g testing medium and covered with a piece of wet paper under the lid. Seven days later, mortality was recorded. The leaves of cabbage (for P. xylostella) and rape (for C. bowringi) were cut into 6-cm disks, dipped into Cry64Ba/Cry64Ca solution for 10 s, air dried, and placed onto a plastic petri dish with a wet filter on the bottom to maintain humidity. Ten larvae were placed on each disk. Mortality was recorded at 48 h. All tested insects were reared at 27°C. Protein buffer (Tris-HCl at 20 mmol/liter; pH 8.0) was used as a negative control. Bioassays were repeated three times. The LC₅₀ and 95% confidence limits (CL) were calculated by SPSS 21.0 with probit analysis.

Binding assay of Cry64Ba/Cry64Ca with BBMV.The whole bodies of 3rd instar L. striatellus nymphs were used for BBMV isolation by the differential precipitation method using MgCl₂ as reported (42). Quantification of total BBMV protein was done by the Bradford method (43). Cry64Ba/Cry64Ca protein (1 mg) was labeled with fluorescein DyLight 488 N-hydroxysuccinimide ester according to the manufacturer's instructions (Thermo Fisher, USA). Different concentrations (from 0 to 900 nmol/liter) of Cry64Ba/Cry64Ca were added to a mixture of 10 μg BBMV in binding buffer (0.1% BSA, 0.1% Tween 20 in phosphate-buffered saline [PBS]) in a 100-μl final volume and incubated for 1 h at room temperature. Elimination of unbound protein and detection of the amount of binding protein were done as reported by Jiang et al. (44). Briefly, after washing out the unbound protein by centrifugation, pellets containing the BBMV with the bound toxins were suspended in Laemmli sample buffer and analyzed by SDS-PAGE. The gel was scanned to collect green fluorescence signal (488 nm excitation [Ex]/518 nm emission [Em]) in a Typhoon 9410 scanner (GE Healthcare). The relative binding capacity is defined as the ratio of the fluorescence densitometry of binding protein to that of 50 ng labeled Cry64Ba/Cry64Ca. The fluorescence densitometry was read by ImageJ software (http://imagej.nih.gov/ij/) on the scanned image. The homologous competition binding assay was performed as follows: first 0 to 20,000 nmol/liter unlabeled Cry64Ba/Cry64Ca or trypsin-digested Cry1Ac protein was incubated with BBMV for 30 min as previously described, then 50 nmol/liter fluorescein-labeled Cry64Ba/Cry64Ca protein was added and incubated for 1 h. The sample was processed as described above. All of the binding experiments were repeated three times.

Histopathological examination of L. striatellus midgut.After 48, 72, and 96 h of toxin exposure of L. striatellus to Cry64Ba/Cry64Ca at a final concentration of 5 μg/ml, the midgut tissue was dissected and fixed in 2.5% glutaraldehyde in phosphate buffer (PB) (0.1 mol/liter, pH 7.2) at 4°C overnight. The fixed midguts were washed with PB three times and then postfixed with 1% osmium tetroxide for 1.5 h. Samples were washed and dehydrated in graded acetone and embedded in Spurr embedding resin. Samples were sectioned at a 100-nm thickness and stained with uranyl acetate and lead citrate. Finally, the sections were examined with an HT-7700 electron microscope (Hitachi, Japan).

Cytotoxicity assay.HepG2 cells were cultured and used to test the cytotoxicity of Cry64Ba/Cry64Ca by GenScript (Nanjing, China). Cytotoxicity assays were performed in 384-well microtest plates. Each well contained 40 μl cell suspension including 800 cells. Cry64Ba/Cry64Ca was added in 2-fold serial dilutions with a highest final concentration of 10 μg/ml. After 72 h of incubation, 30 μl CellTiter-Glo (Promega, USA) was added to each well, followed by further incubation for 2 to 3 min at room temperature. Finally, the number of relative light units (RLU) was measured on a PHERAstar microplate reader (BMG Labtech, Germany). Tris-HCl (20 mmol/liter, pH 8.0) was used as a negative control (NC). Doxorubicin (10 μmol/liter) was used as a positive control. Cell growth inhibition was calculated as follows: % of cell growth inhibition = 100% × (1 − RLU_Sample/RLU_NC).

Hemolysis assays.Cry64Ba/Cry64Ca protein (from 6.25 to 100 μg/ml) dissolved in PBS was mixed with 50 μl of 2% sheep erythrocyte suspension in 0.9% NaCl, and the final volume was fixed to 200 μl. After 1 h of incubation at room temperature, the cells were centrifuged at 1,000 × g for 5 min, 150 μl of the supernatant was collected, and the absorbance (Ab) was measured at 540 nm in a FlexStation 3 microplate reader (Molecular Devices, USA). As the negative control (NC), 0.9% NaCl was used while the positive control (PC) corresponding to 100% hemolysis contained 1% Triton X-100. Percent hemolysis was calculated as follows: (Ab_sample−Ab_NC)/(Ab_PC−Ab_NC) × 100%.

Accession number(s).Draft genome sequences of Bt 1012 have been deposited in GenBank under accession no. NTGL00000000. The nucleotide acid sequence data for cry64Ba and cry64Ca are available in GenBank under accession no. KC960014.