Knockdown of CYP301B1 and CYP6AX1v2 increases the susceptibility of the brown planthopper to beta-asarone, a potential plant-derived insecticide.

The cytochrome P450 monooxygenases of insects play crucial roles in the metabolic detoxification of insecticides. Our previous finding showed that two cytochrome P450 genes, both CYP301B1 and CYP6AX1v2, in the BPH underwent overexpression due to ß-asarone. In this study, we investigated the molecular characteristics, expression patterns and functions of these two cytochrome P450 genes. The results showed that CYP301B1 had the highest expression level in the eggs, while CYP6AX1v2 was expressed in macropterous female adults. Moreover, the expression level of CYP301B1 in the head was higher than that in the integument, fat body and gut. The expression level of CYP6AX1v2 in the fat body and gut was higher than that in head and integument. Importantly, silencing CYP301B1 and CYP6AX1v2 separately could increase the sensitivity, resulting in significant higher mortality of BPH following treatment with ß-asarone. Our findings indicated that CYP301B1 and CYP6AX1v2 could contribute to the resistance of BPH to ß-asarone, and these two genes may be involved in the detoxification metabolism of ß-asarone in BPH.

Candidate detoxification-related genes in brown planthopper, Nilaparvata lugens, in response to ß-asarone based on transcriptomic analysis.

Nilaparvata lugens(Stål) is a serious pest of rice and has evolved different levels of resistance against most chemical pesticides. ß-asarone is the main bioactive insecticidal compound of Acorus calamus L. that shows strong insecticidal activity against pests. In this study, we conducted a bioassay experiment to determine the contact toxicity of ß-asarone to N. lugens nymphs. The LD30 sublethal dose was 0.106 µg per nymph, with 95% confidence limits of 0.070-0.140 µg. We applied the LD30 concentration of ß-asarone to nymphs for 24 h or 72 h and then performed a transcriptome sequence analysis by referencing the N. lugens genome to characterize the variation. The transcriptomic analysis showed that several GO terms and KEGG pathways presented significant changes. Individually, 126 differentially expressed genes (DEGs), including 72 upregulated and 54 downregulated genes, were identified at 24 h, and 1771 DEGs, including 882 upregulated and 889 downregulated genes, were identified at 72 h. From the DEGs, we identified a total of 40 detoxification-related genes, including eighteen Cytochrome P450 monooxygenase genes (P450s), three Glutathione S-transferase genes, one Carboxylesterase gene, twelve UDP-glucosyltransferases and six ATP-binding cassette genes. We selected the eighteen P450s for subsequent verification by quantitative PCR. These findings indicated that ß-asarone presented strong contact toxicity to N. lugens nymphs and induced obvious variation of detoxification-related genes that may be involved in the response to ß-asarone.

Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1).

Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control.

Improvement of crystal solubility and increasing toxicity against Caenorhabditis elegans by asparagine substitution in block 3 of Bacillus thuringiensis crystal protein Cry5Ba.

The crystal proteins from Bacillus thuringiensis are widely used for their specific toxicity against insects and nematodes. The highly conserved sequence blocks play an important role in Cry protein stability and flexibility, the basis of toxicity. The block 3 in Cry5Ba subfamily has a shorter sequence (only 12 residues) and more asparagine residues than that of others which harbor about 48 residues but only one asparagine. Based on the theoretical structure model of Cry5Ba, all three asparagines in block 3 are closely located in the interface of putative three domains, implying their probable importance in structure and function. In this study, all three asparagines in Cry5Ba2 block 3 were individually substituted with alanine by site-directed mutagenesis. The wild-type and mutant proteins were overexpressed and crystallized in acrystalliferous B. thuringiensis strain BMB171. However, the crystals formed in one of the mutants, designated N586A, abnormally disappeared and dissolved into the culture supernatant once the sporulation cells lysed, whereas the Cry5Ba crystal and the other mutant crystals were stable. The mutant N586A crystal, isolated from sporulation cells by the ultrasonic process, was found to be easily dissolved at wide range of pH value (5.0 to 10.0). Moreover, the toxicity assays showed that the mutant N586A exhibited nearly 9-fold-higher activity against nematodes and damaged the host's intestine more efficiently than the native Cry5Ba2. These data support the presumption that the amide residue Asn586 at the interface of domains might adversely affect the protein flexibility, solubility and resultant toxicity of Cry5Ba.

Synergistic activity between Bacillus thuringiensis Cry6Aa and Cry55Aa toxins against Meloidogyne incognita.

Plant-parasitic nematodes are the most destructive group of plant pathogens worldwide and are extremely challenging to control. Some Bacillus thuringiensis crystal proteins are highly toxic to the plant-parasitic nematode Meloidogyne incognita. In this study, the nematicidal crystal proteins Cry6Aa, Cry5Ba and Cry55Aa were tested against M. incognita to select the best toxin combination for its management. The results showed that a combination of Cry6Aa and Cry55Aa showed significant synergistic toxicity against M. incognita, and the highest synergistic effect (five times the expected toxicity of the two toxins calculated from their separate toxicities) was observed when they were combined in a 1:1 ratio. Furthermore, ligand blot analyses of the interaction between total proteins of M. incognita and the three toxins showed many different signal bands, indicating that there is a range of host proteins with which the toxins can interact. One explanation of the observed synergism is that the toxins damage the host in diverse ways, and they may thus act cooperatively and thereby show greater toxicity in combination. Our discovery provides an effective strategy for controlling M. incognita by using a combination of Cry6Aa and Cry55Aa.

Single cysteine substitution in Bacillus thuringiensis Cry7Ba1 improves the crystal solubility and produces toxicity to Plutella xylostella larvae.

Many Bacillus thuringiensis isolates have no demonstrated toxicity against insects. In this study, a novel holotype crystal protein gene cry7Ba1 was isolated from a 'non-insecticidal'B. thuringiensis strain YBT-978. The Cry7Ba1 protein showed high toxicity against Plutella xylostella larvae after the crystals were dissolved at pH 12.5, suggesting that the 'non-insecticidal' properties of this protein were due to insolubility in the normal insect midgut pH environment. After the C-terminal half of Cry7Ba1 was replaced by that of Cry1Ac or Cry1C proteins, the recombinant protein inclusions could be dissolved at pH 9.5, and exhibited high toxicity against P. xylostella larvae. This result proved the insolubility of Cry7Ba1 crystal was determined by the structure of its C-terminal half. Further, six mutations were constructed by substituting cysteine residues with serine. Solubility studies showed that the crystals from mutants C697S, C834S and C854S could be dissolved at lower pH (10.5, 9.5 and 11.5 respectively). Bioassays showed that crystals from mutant C834S were toxic to P. xylostella larvae. Our discoveries suggest that a single cysteine residue located in the C-terminal half of the protein determines the solubility and toxicity of some nontoxic crystal proteins. This study provides a strategy to isolate novel insecticidal crystal protein genes from 'non-insecticidal'B. thuringiensis strains.