Abstract
Nematodes are known to be harmful to various crops, vegetables, plants and insects. The present study reports that, chitin upregulates the activity of chitinase (20%) and nematicidal potential (15%) of Pseudomonas aeruginosa. The chitinase gene (pachi) from P. aeruginosa was cloned and its nematicidal activity of pachi protein against Caenorhabditis elegans was studied. The mortality rate induced by pachi increased by 6.3-fold when in association with Cry21Aa from Bacillus thuringiensis. Pachi efficiently killed C. elegans in its native state (LC50 = 387.3 ± 31.7 μg/ml), as well as in association with Cry21Aa (LC50 = 30.9 ± 4.1 μg/ml), by degrading the cuticle, egg shell and intestine in a relatively short time period of 24 h. To explore the nematidal potential of chitinase, six fusion proteins were constructed using gene engineering techniques. The CHACry showed higher activity against C. elegans than others owing to its high solubility. Notably, the CHACry showed a synergistic factor of 4.1 versus 3.5 a mixture [1:1] of pachi and Cry21Aa. The present study has identified eco-friendly biological routes (e.g., mixed proteins, fusion proteins) with potent nematicidal activity, which not only can help to prevent major crop losses but also strengthen the agro-economy and increase gross crop yield.
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Introduction
Nematodes infect the root, stem and leaves of cultural crops, leading to plant wilting, chlorosis, reduced growth and ultimately death, resulting in major economic losses. In order to control nematodes, various chemicals have been used worldwide. These chemicals not only are toxic to the plants but also have various harmful effects on human health and the environment1. Gene engineering has opened new gateways to develop nontoxic bio-control agents that are effective in protecting from nematodiasis2. In the study of bio-control of nematodiasis, C. elegans has been used widely as an important model organism owing to its genetic manipulability and short life cycle3,4. A literature survey revealed that some nematophagous bacteria were effective in killing nematodes. For example, Bacillus thuringiensis, Pseudomonas and Xenorhabdus nematohilus, found in the rhizosphere of plants, showed high nematicidal activity12 and Clonostachys rosea13, which killed nematodes via egg shell digestion and cuticle hydrolysis7. As chitin was the main component of the egg shell and cuticle of nematodes and acted as a target for these nematicidal factors14, chitin and chitosan were incorporated into the soil to reduce nematode infection by inducing rhizobacteria to produce chitinase15. Pseudomonas was an important rhizobacteria strain, which could produce some hydrolases (protease and chitinase) in killing nematodes16,17,Protein expression and purification E. coli BL21 (DE3) cells harboring pGEX-6p-pachi, named DE3/pGEX-6p-pachi, were inoculated into LB broth (ampicillin; 100 μg/ml) with shaking at 37 °C for 12 h. The seed culture was used to inoculate production broth (v/v, 2/100) and growth was induced by adding IPTG (1 mM) after 2–3 h. The IPTG-induced production broth was incubated at 18 °C for 14 h with shaking (250 rpm/min). Finally, the induced cells were collected by centrifugation, resuspended in phosphate-buffered saline (PBS) buffer (NaCl 0.8%, KCl 0.02%, Na2HPO4 0.14%, KH2PO4 0.03%; pH 7.0) and homogenized using a high-pressure homogenizer (NS100IL 2K, Niro Soavi, Germany). The target proteins (Cry21Aa, pachi and CHACry) were purified using a glutathione S-transferase (GST) Gene Fusion System (GE Healthcare, USA) and eluted from the GST tag by 3C proteases (PreScission, Pharmacia). The molecular weight was analyzed by SDS-PAGE with 10% polyacrylamide gels. The protein concentration was measured by the Bradford method using bovine serum albumin (BSA) as a standard. The purified proteins were used for bioassays including quantitative mortality tests, brood size assays and growth analyses. The bioassay procedures and 50% lethal/inhibition/growth concentration (LC50/IC50/GC50) evaluations were undertaken according to the method of Bischof et al.52. The cuticle was separated from the nematode body as described by Cox et al.14. Cuticles (eggs) was incubated with pachi at 20 °C and 37 °C for various time periods. The L4 worms were incubated with pachi, Cry21Aa and the CHACry fusion protein at 20 °C and 37 °C for various time periods. Pachi was used to study complete digestion at 1 mg/ml for the cuticle and at 200 μg/ml for the egg shell and intestine. The effects of Cry21Aa on the intestine were assessed at a final concentration of 150 μg/ml, while CHACry was assessed at 100 μg/ml. Pachi (1 mg/ml) was used as an effective nematicidal dose to ensure the complete killing of C. elegans. The synergistic factor was calculated by the formula of Tabashnik et al.53: 1/LC50(m) = Ra/LC50(a) + Rb/LC50(b), where Ra and Rb indicate the percentage of toxin A and toxin B proteins used in the final mixture; LC50(a) and LC50(b) represent the LC50 values for each toxin and LC50(m) is the expected theoretical value of LC50 calculated from the formula above. The real LC50 value was calculated from the bioassay for the observed toxicity of the mixture. Synergism was indicated with a SF value of greater than 1. Independent experiments were repeated at least three times. All of the data obtained were analyzed using GraphPad Prism 5.0 and Excel 2003 software for figures and LC50 values. All of the values were expressed as the mean values ± standard deviation, with the statistical significance set at p < 0.05. To study the effect of each protein on nematodes and their body parts, images were captured using a 20X objective lens with confocal laser scanning microscopy on a Zeiss LSM 510 microscope (CLSM; Zeiss LSM 510) imaging. Mixture of diethyl ether/ethanol absolute (1:1) was used as anaesthetic treatment to keep worms static during image capture. All pictures were processed using Photoshop 7.0 software and the worm sizes were calculated using ImageJ 2.4.1.7. The nucleotide sequences of Pseudomonas aeruginosa 16S and pachi have been submitted to GeneBank under accession numbers KR007310 (16S) and KR007311 (pachi).Quantitative analysis of nematicidal activity
Cuticle and egg shell digestion
Synergistic activity assays for pachi and Cry21Aa in L4 worms of the N2 strain
Statistical analysis
Image analysis by confocal microscopy
Nucleotide sequence accession number
Additional Information
How to cite this article: Chen, L. et al. Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa. Sci. Rep. 5, 14395; doi: 10.1038/srep14395 (2015).
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Acknowledgements
This work was supported by grants from National Basic Research Program (973) of China (2013CB127504) and the China 948 Program of Ministry of Agriculture (2011-G25).
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L.C. designed the experiments and wrote the manuscript. L.C. and J.C. performed the experiments. L.C. analyzed the data. H.J., Q.C., J.C., A.K., G.W., Z.L. and M.S. contributed reagents/materials/analysis tools.
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Chen, L., Jiang, H., Cheng, Q. et al. Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa. Sci Rep 5, 14395 (2015). https://doi.org/10.1038/srep14395
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DOI: https://doi.org/10.1038/srep14395
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