A Novel Protein Elicitor PeBL2, from Brevibacillus laterosporus A60, Induces Systemic Resistance against Botrytis cinerea in Tobacco Plant

Abstract

Here, we reported a novel secreted protein elicitor PeBL2 from Brevibacillus laterosporus A60, which can induce hypersensitive response in tobacco (Nicotiana benthamiana). The ion-exchange chromatography, high-performance liquid chromatography (HPLC) and mass spectrometry were performed for identification of protein elicitor. The 471 bp PeBL2 gene produces a 17.22 kDa protein with 156 amino acids containing an 84-residue signal peptide. Consistent with endogenous protein, the recombinant protein expressed in Escherichia coli induced the typical hypersensitive response (HR) and necrosis in tobacco leaves. Additionally, PeBL2 also triggered early defensive response of generation of reactive oxygen species ($H_2O_2$ and $O_2{^-}$) and systemic resistance against of B. cinerea. Our findings shed new light on a novel strategy for biocontrol using B. laterosporus A60.

Fig. 4. Phylogenetic tree based on 16S rRNA sequencing, which was generated using the neighbour-joining method. Relationships of. ERM16658.1. sequence (highlighted in yellow) to other Brevibacillus species are shown. Bar, 1 change per nucleotide position.

Fig. 6. ROS burst in tobacco cells after PeBL2 treatment. The brown DAB-stained precipitates represent ROS burst. (A) No ROS generate in tobacco leaves treated with BSA (control). (B) Significant ROS were observed in the recombinant PeBL2-treated areas.

Fig. 7. Induced disease resistance against Botrytis cinerea in tobacco. (A) Representative phenotypes of disease caused by B. cinerea in PeBL2 and BSA-infiltrated tobacco leaves. The sizes of the lesions caused by B. cinerea in PeBL2-treated leaves were smaller than that in BSA at 36 h post-infiltration. (B) Lesion sizes caused by B. cinerea were measured in leaves with PeBL2 or BSA-treated plants. Data presented in (B) were calculated as follows: Inhibition % = [(No (size) of lesions on control leaves − No (size) of lesions on proteintreated leaves)/No (size) of lesions on control leaves] × 100%. (B1) data of treated and untreated plants were plotted showing inhibition percentage rate. (B2) Statistical analysis was performed using Student’s t-test (Circles), Box Plots for BSA (control) and PEBL2 (treated) representing differences among both treatments indicate significant differences between PeBL2 and BSA treatment.

Fig. 1. Purification of the PeBL2 protein. (A) Cation exchange chromatography using a HisTrap SP HP column (2.5 cm × 20 cm). The column was washed with buffer A (25 mM MES-NaOH, pH 6.2) to remove any unbound proteins, and the bound proteins were eluted with a linear gradient of increasing NaCl (0-1 M). Buffer B (25 mM Tris, 200 mM NaCl, 500 mM imidazole, pH 8.0) was used to B. Two fractions (peak a and peak b) were produced. (B) Fractions (peak a and peak b) along with BSA (control) included in the test for HR. Out of these fractions peak a showing HR in tobacco leaves. (C) SDS-PAGE of crude proteins.

Fig. 2. Purification of the active fraction. (A) Waters Atlantis T3 C18 reversed-phase column (2.1 mm × 150 mm, 3.5 m, 40℃) was equilibrated with 5% CAN and acetonitrile/2 mM NH4FA/0.1% FA/water. The concentration of ACN in the eluted solvent was raised from 10% (v/v) to 60% (v/v) over 28 min using a linear gradient at a flow rate of 0.2 ml/min. The peaks a, b, c, d, e and f were produced by HPLC. (B) All peaks were checked for HR in tobacco leaves. Out of all peaks peak d displayed an obvious HR.

Fig. 3. Further purification of the active protein with HPLC (A) HPLC was used to further purify fraction D using Zorbax-Eclipse (XDB-C18). (B) After purification, fraction D was checked by Tricine SDS-PAGE to determine the size of the protein, which is shown as about 17 kDa. (C) The HR activation of purified single protein was checked as compared to BSA.

Fig. 5. Expression and purification of protein PeBL2 in E. coli. (A) PCR was used to amplify the full-length DNA sequence encoding PeBL2 from the B. laterosporus A60 strain. The length of the PeBL2 gene is 471 bp, which encodes a protein of 156 amino acids with a theoretical molecular weight of 17 kDa. The PeBL2 gene was cloned and then ligated into pET28a. (B) The recombinant protein PeBL2 was expressed and purified. SDS-PAGE of the purified elicitor protein, PeBL2, displaying a single band by Coomassie Brilliant Blue R-250 staining. (C) The hypersensitive response induced by the recombinant elicitor protein, PeBL2. The response was observed at 24 h post-infiltration. The elicitor treatment (50 μm) and control treatment with BSA are shown.