Insecticidal Activity of Chitinases from Xenorhabdus nematophila HB310 and Its Relationship with the Toxin Complex.

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex (Tc). The chi60 and chi70 chitinase genes are located on the gene cluster encoding Tc toxins. To clarify the insecticidal activity of chitinases and their relationship with Tc toxins, the insecticidal activity of the chitinases was assessed on Helicoverpa armigera. Then, the chi60 and chi70 genes of X. nematophila HB310 were knocked out by the pJQ200SK suicide plasmid knockout system. The insecticidal activity of Tc toxin from the wild-type strain (WT) and mutant strains was carried out. The results demonstrate that Chi60 and Chi70 had an obvious growth inhibition effect against the second instar larvae of H. armigera with growth-inhibiting rates of 81.99% and 90.51%, respectively. Chi70 had a synergistic effect with the insecticidal toxicity of Tc toxins, but Chi60 had no synergistic effect with Tc toxins. After feeding Chi60 and Chi70, the peritrophic membrane of H. armigera became inelastic, was easily broken and leaked blue dextran. The Δchi60, Δchi70 and Δchi60-chi70 mutant strains were successfully screened. The toxicity of Tc toxins from the WT, Δchi60, Δchi70 and Δchi60-chi70 was 196.11 µg/mL, 757.25 µg/mL, 885.74 µg/mL and 20,049.83 µg/mL, respectively. The insecticidal activity of Tc toxins from Δchi60 and Δchi70 was 3.861 and 4.517 times lower than that of Tc toxins from the WT, respectively, while the insecticidal activity of Tc toxins from the Δchi60-chi70 mutant strain almost disappeared. These results indicate that the presence of chi60 and chi70 is indispensable for the toxicity of Tc toxins.

Functional characterization of a novel, highly expressed ion-driven sugar antiporter in the thoracic muscles of Helicoverpa armigera.

Sugar transporters (STs), which mainly mediate cellular sugar exchanges, play critical physiological roles in living organisms, and they may be responsible for sugar exchanges among various insect tissues. However, the molecular and physiological functions of insect STs are largely unknown. Here, 16 STs of Helicoverpa armigera were identified. A phylogenetic analysis classified the putative HaSTs into 12 sub-families, and those identified in this study were distributed into 6 sub-families. Real-time polymerase chain reaction indicated that the 16 HaSTs had diverse tissue-specific expression levels. One transporter, HaST10, was highly expressed in thoracic muscles. A functional study using a Xenopus oocyte expression system revealed that HaST10 mediated both H+ -driven trehalose and Na+ -driven glucose antiport activities with high transport efficiency and low affinity levels. A HaST10 knockout clearly impaired the performance of H. armigera. Thus, HaST10 may participate in sugar-supply regulation and have essential physiological roles in H. armigera.

Identification of Arylphorin interacting with the insecticidal protein PirAB from Xenorhabdus nematophila by yeast two-hybrid system.

PirAB toxin was initially found in the Photorhabdus luminescens TT01 strain and is a demonstrated binary toxin with high insecticidal activity. In this paper, we co-expressed the pirAB gene of Xenorhabdus nematophila HB310 in a prokaryotic expression system, and we found that the PirAB protein showed high hemocoel insecticidal activity against Galleria mellonella, Helicoverpa armigera and Spodoptera exigua. LD50 values were 1.562, 2.003 and 2.17 µg/larvae for G. mellonella, H. armigera, and S. exigua, respectively (p > 0.05). Additionally, PirAB-interaction proteins were identified from G. mellonella by 6 × His Protein Pulldown combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of which, arylphorin of G. mellonella showed the highest matching rate. A protein domain conservative structure analysis indicated that arylphorin has three domains including Hemocyanin-N, Hemocyanin-M, and Hemocyanin-C. Among these protein domains, Hemocyanin-C has immune and recognition functions. Further, Hemocyanin-C domain of arylphorin was identified to interact with PirA but not PirB by Yeast two-hybrid system. These findings reveal, for the first time, new host protein interacting with PirAB. The identification of interaction protein may serve as the foundation for further study on the function and insecticidal mechanism of this binary toxin from Xenorhabdus.

Expression and characterization of two chitinases with synergistic effect and antifungal activity from Xenorhabdus nematophila.

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.

Characteristics and Function of the Chitin Binding Protein from Xenorhabdus nematophila.

BACKGROUND: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). OBJECTIVE: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. METHODS: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. RESULTS: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. CONCLUSION: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.

PirAB protein from Xenorhabdus nematophila HB310 exhibits a binary toxin with insecticidal activity and cytotoxicity in Galleria mellonella.

PirAB (Photorhabdus insect-related proteins, PirAB) toxin was initially found in the Photorhabdus luminescens TT01 strain and has been shown to be a binary toxin with high insecticidal activity. Based on GenBank data, this gene was also found in the Xenorhabdus nematophila genome sequence. The predicted amino acid sequence of pirA and pirB in the genome of X. nematophila showed 51% and 50% identity with those gene sequences from P. luminescens. The purpose of this experiment is to identify the relevant information for this toxin gene in X. nematophila. The pirA, pirB and pirAB genes of X. nematophila HB310 were cloned and expressed in Escherichia coli BL21 (DE3) using the pET-28a vector. A PirAB-fusion protein (PirAB-F) was constructed by linking the pirA and pirB genes with the flexible linker (Gly)4 DNA encoding sequence and then efficiently expressed in E. coli. The hemocoel and oral insecticidal activities of the recombinant proteins were analyzed against the larvae of Galleria mellonella. The results show that PirA/B alone, PirA/B mixture, co-expressed PirAB protein, and PirAB-F all had no oral insecticidal activity against the second-instar larvae of G. mellonella. Only PirA/B mixture and co-expressed PirAB protein had hemocoel insecticidal activity against G. mellonella fifth-instar larvae, with an LD50 of 2.718µg/larva or 1.566µg/larva, respectively. Therefore, we confirmed that PirAB protein of X. nematophila HB310 is a binary insecticidal toxin. The successful expression and purification of PirAB laid a foundation for further studies on the function, insecticidal mechanism and expression regulation of the binary toxin.

Aminopeptidase N5 (APN5) as a Putative Functional Receptor of Cry1Ac Toxin in the Larvae of Athetis lepigone.

Athetis lepigone was a new lepidopteran pest and caused severe damage to maize crops in China. We have detected that Cry1Ac protoxin and toxin were highly active against the larvae of A. lepigone. However, there is no report about the mode of action of Bt Cry1Ac toxin against this pest until now. A 110 kDa APN5 protein from BBMV of A. lepigone was identified as the binding receptor of Cry1Ac toxin using Ligand blotting. The Cry1Ac receptor APN5 was cloned from A. lepigone larval midgut mRNA and named as AlAPN5 (GenBank accession no.: KU950745). AlAPN5 had a GATEN motif and been classified to Class 5 APNs. 79.2% reduction in mortality was observed when A. lepigone larvae were injected with siRNA of the AlAPN5 gene and treated with Cry1Ac toxin. These data demonstrate that AlAPN5 is a putative functional receptor and maybe the only receptor of Cry1Ac in A. lepigone.

Detection of minimum activity fragment of a novel Cry7Ab3 protein specific to Henosepilachna vigintioctomaculata (Coleopteran: Coccinellidae).

Bacillus thuringiensis Cry7Ab3 toxin has insecticidal activity against larvae of Henosepilachna vigintioctomaculata. Cry7Ab3 toxin is solubilized under alkaline condition and activated by proteases within the larval gut. In order to assess the functions of the N- and C-terminal regions, several N- and C-terminal truncated forms of Cry7Ab3 were constructed. It was determined that amino acid removal at the N-terminal, which disrupt the α-helico structure, resulted in the inactivation of the protein. The deletion of 512 amino acids from the C-terminus reduced the toxicity. However, the deletion of 481 amino acids from the C-terminus resulted in the highest activity. These findings directly demonstrated the critical roles of N- and C-terminal amino acids on the toxicity of Cry7Ab3.

Identification of Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) midgut putative receptor for Bacillus thuringiensis insecticidal Cry7Ab3 toxin.

Bt WZ-9 strain, containing a single Cry7Ab3 toxin, had effective insecticidal activity against larvae of Henosepilachna vigintioctomaculata. By incubation with larvae midgut homogenate and trypsin in vitro, 130 kDa Cry7Ab3 protoxin was degraded into the ∼75 kDa proteinase-resistant fragments. In vivo analysis, 130 kDa Cry7Ab3 protoxin was also processed into ∼75 kDa fragment. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae causes acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. A ligand blotting experiment demonstrated that Cry7Ab3 toxin bound a 220 kDa BBMV protein. This receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). The cadherin protein may be the receptor of Cry7Ab3. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae.

[Purification and characterization of the haemocoel insecticide Tp40 from Xenorhabdus nematophila].

OBJECTIVE: Xenorhabdus nematophila is an insect pathogen bacterium symbiotically associated with entomopathogenic nematode. The bacteria produce a number of toxins to overcome immune response of insect hosts and kill their hosts. We purified a novel haemocoel insecticidal protein from X. nematophila HB310, cloned and analysed gene sequence of this novel protein. METHODS: We isolated and purified the insecticidal protein by methods of salting out and native-PAGE from the intracellular proteins of X. nematophila HB310. We tested the virulence of the protein by direct injection into fifth-instar Galleria mellonella larvae. The protein was identified by western blotting. The gene of insecticidal protein was cloned by PCR and analyzed in GenBank. RESULTS: We purified a novel haemocoel insecticidal protein that was named as Tp40. The injectable hemocoelic potency (LD50) of Tp40 was 68.54 ng/larva against fifth-instar G. mellonella larvae. The SDS-PAGE spectrum of Tp40 only showed a single - 42kDa band. Western blotting with an antibody that was highly specific to the known Txp40 indicated that Tp40 was homologous to the known Txp40 and only existed inside cells. The nucleotide sequences of tp40 gene have been deposited in GenBank (accession number GenBank: EU095326). The size of the open reading frame of tp40 was 1107bp, encoding a peptide of 368 amino acid residues, with a theoretical molecular weight 41.5 kDa and an isoelectricpoint (IP) 8.66. The Tp40 shared 85%-99% homology of nucleotide sequences and 70%-99% amino acids with those of 13 group strains. CONCLUSION: Tp40 is high injectable virulent for G. mellonella larvae and its gene/protein sequence is highly conserved, which play a key role during the bacterium-nematode killing host insects process.