Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa

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.

Figure 1. Chitinase as the potential toxin of P. aeruginosa against C. elegans.

(A) Effects of different temperatures on nematicidal activity P. aeruginosa. (B) Effects of different acidic and alkaline conditions on nematicidal activity of P. aeruginosa. (C) Effects of chitin on the chitinase activity and nematicidal activity of P. aeruginosa. The concentration of N-acetylglucosamine (NAG) indicated chitinase activity. Nematicidal activity was measured using L4 worms (~80 worms each well) at 20 °C.

Figure 2. Phylogenetic analysis of the relationships among chitinases from different bacteria.

The phylogenetic tree was constructed using the neighbor-joining method (MEGA6.0). All chitinase sequences were obtained from GeneBank and PDB (http://www.rcsb.org/pdb/home/home.do), and the accession numbers of chitinase were listed in the form of “AAM48520”, except for pachi. These chitinases were reported to infect multiple phytopathogenic organisms such as fungi, insects, and nematodes.

Figure 3. Schematic representation of construction, expression and nematicidal activity of fusion proteins.

(A) Fusion protein construction. (B) Fusion protein tested on C. elegans. (C) Fusion protein expression. Lane M: Standard protein molecular mass marker (10–225.0 kDa). Lane 2–7: Induced cell lysate supernatants of pGEX-6p-1, pGEX-6p-Cry21Aapachi, pGEX-6p-pachiCry21Aa, pGEX-6p-Crypachi, pGEX-6p-pachiCry, pGEX-6p-CryCHA, and pGEX-6p-CHACry. The arrows point to target proteins.

Figure 4. Recombinant plasmid construction and SDS-PAGE analysis of protein expression.

(A) Fusion protein construction and partial restriction enzymatic site. (B) SDS-PAGE analysis of recombination protein expression in E. coli and purification. The arrows point to target proteins. Lane 1: The standard protein molecular mass marker (14.4–116.0 kDa). Lane 2–5: Induced cell lysate supernatants of pGEX-6p-1, pGEX-6p-CHACry, pGEX-6p-Cry21Aa, and pGEX-6p-pachi harbored in E. coli. Lane 6-8: Induced cell lysate sediments of pGEX-6p-CHACry, pGEX-6p-Cry21Aa, and pGEX-6p-pachi harbored in E. coli. Lane 9–11: Purified CHACry, Cry21Aa, and pachi proteins.

Figure 5. Pachi enhances Cry21Aa activity against C. elegans.

(A) Mortality analysis was performed on L4 worms. (B) Brood size tests were performed on L4 worms. (C) Growth assays were performed on L1 larva. Error bars represent the standard deviation from the average values within three parallel experiments; (D) The effects of growth inhibition. All images were captured at 10X magnification under a Zeiss LSM 510 confocal microscope.

Figure 6. Egg shell and cuticle digestion by pachi.

(A) Eggs were incubated with PBS; (B) Eggs were incubated with pachi; (C) Cuticles were incubated with PBS; (D) Cuticles were incubated with pachi. Images were taken under a Zeiss LSM 510 confocal microscope at 20X magnification.

Figure 7. Effects of Cry21Aa, pachi, and CHACry on the intestine.

(A) Worms were incubated with PBS; (B) Worms were incubated with Cry21Aa; (C) Worms were incubated with pachi; (D) Worms were incubated with CHACry. Images were taken under a Zeiss LSM 510 confocal microscope at 20X magnification.