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

Abstract

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 Comparison of Three Chitinases from Symbiotic Bacteria of Entomopathogenic Nematodes

“…In particular, XnChi is relatively close to the X. nematophila ATCC19061 chitinase (Genbank accession number: WP 010846090.1), which shows 99.69% homology to Chi70 (Genbank accession number: GK44779.1) of X. nematophila . Chi70 was reported to have insecticidal and antifungal activities against certain pests and fungi, and to improve insecticidal activity of BtCryAc and Tc toxins [ 23 , 31 , 32 ]. Thus, symbiotic bacterial chitinases of EPNs could be promising control agents of insect pests and plant pathogenic fungi by playing an important role in pesticidal activity.…”

“…Thus, chitinase’s key enzyme has received increasing attention as biopesticide for the control of insect pests and fungal diseases because chitin synthesis is performed using a range of organisms, including fungi and insects, and key enzyme in biosynthesis [ 21 , 22 ]. Chitinase is a Glycoside hydrolase (GH) that acts to degrade chitin using hydrolyzing glycosidic bonds [ 23 ], and it is classified into two main groups, chitinase (EC3.2.1.14) and β-acetylhexosaminidase (EC 3.2.1.52), officially called endochitinase and exochitinase, respectively, depending on the products produced during the hydrolysis process [ 24 , 25 ]. Endochitinase cleaves into the chitin chain at an internal site.…”

“…In particular, as chitinases belong to the GH18 family, they are widely distributed in almost all organisms and were found to be involved in many physiological processes including tissue degradation, developmental regulation, pathogenicity, and immune defense [ 28 ]. These chitinases from a variety of microorganisms which facilitate the invasion of pathogens by causing structural changes in the peritrophic membrane of insects, which is primarily composed of chitin, promoting the accessibility of the substrate for the pathogen into the haemocoel, and leading to the interception of nutrient absorption in the midgut [ 23 , 29 ]. Furthermore, chitinases can facilitate the binding process of toxins to specific receptors in the midgut epithelium of insects and enhance the insecticidal activity of entomopathogenic bacteria [ 30 ].…”

“…In bacterium Yersinia entomophaga MH96 isolated from diseased grass grub larva of Costelytra zealandica (Coleoptera: Scarabaeidae), the 3D structure of Tc toxins includes two putative chitinases (Chi1 and Chi2) which are essential for complex formation [ 31 ]. In addition, two chitinase (chi60 and chi70) genes were found in the toxin complex locus of X. nematophila [ 31 ] and the corresponding proteins were shown to be vital for the insecticidal activity against Helicoverpa armigera in the toxicity of Tc toxins [ 23 ]. Moreover, chitinases are able to inhibit the elongation and growth of mycelia and spore germination of fungi [ 32 ], that is, chitinase Chi2A and chitin binding protein (CBP) in the secondary cells of P. luminescens are necessary to inhibit the growth of Fusarium graminearum [ 33 ].…”

Identification and Biocontrol Potential of Entomopathogenic Nematodes and Their Endosymbiotic Bacteria in Apple Orchards against the Codling Moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae)

“…The results of the aforementioned studies indicated that secondary metabolites of different Xenorhabdus and Photohabdus species were able to penetrate through the cuticle and cause remarkable mortalities against tested insects with a varying degree of pathogenicity [44,75,76,83]. Chitinase protein secreted by Xenorhabdus and Photorhabdus bacteria plays a key role in the virulence of symbiotic bacteria by both degrading the cuticles of insects and accelerating the binding process of toxins to target sites [84,85]. The results suggest that toxic metabolites including chitinase protein are present in the cell-free supernatant of symbiotic bacteria and are responsible for mortality.…”