A Novel Protein Elicitor (PeBA1) from Bacillus amyloliquefaciens NC6 Induces Systemic Resistance in Tobacco

Abstract

Here we reported a novel protein elicitor from Bacillus amyloliquefaciens NC6 induced systemic resistance (ISR) in tobacco. The purification was executed by ion-exchange chromatography, native-page extraction and HPLC, and the amino acid sequence was identified by mass spectrometry. This recombinant elicitor protein, expressed in Escherichia coli by an E1 expression vector, had good thermal stability, and the elicitor caused a clearly defined hypersensitive response (HR) necrosis in tobacco leaves. It could also trigger early defence events, including generation of reactive oxygen species (H2O2 and O2 (-)) and phenolic-compound accumulation. Quantitative real-time PCR (Q-RT-PCR) results indicated that several plant defence genes, including the salicylic acid (SA)-responsive PR1a, PR1b, PR5, and phenylalanine ammonia lyase (PAL), as well as the jasmonic acid (JA)-responsive PDF1.2 and CORONATINE INSENSITIVE 1 (COI1), were all up-regulated. Moreover, infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus (TMV) and the fungal pathogen Botrytis cinerea.

Keywords: Hypersensitive response; ROS; induced systemic resistance; protein elicitor.

Fig 1

Purification of PeBA1 from Bacillus amyloliquefaciens NC6. (A) Crude protein from the ammonium sulfate precipitation was purified using the Akta Explorer 10 protein purification system. Using an ion-exchange chromatography column, four peaks (1, 2, 3, and 4) were collected. (B) Peak 2 was further purified by native-PAGE and HPLC. PeBA1 was resolved on an SDS-PAGE gel with a protein molecular mass between 26 and 34 kDa.

Fig 2

Purification of recombinant PeBA1. (A) Total protein expressed in E. coli was purified by the Akta Explorer 10 protein purification system using a His-Trap HP column. Recombinant PeBA1 was eluted with high-concentration imidazole. (B) Expression and purification of recombinant PeBA1. 1: total protein, 2: soluble recombinant PeBA1, 3: filtrate, 4: disrupted cell pellet.

Fig 3

PeBA1-induced HR in plant leaves. (A) HR in tobacco leaves was observed at 24 hours post-infiltration with PeBA1. Red circles were treated with 30 μl PeBA1 (20μM) and showed obvious HR, while buffer (Tris-HCl, pH8.3) as a control (black circles) did not induce HR. (B) RT-PCR was executed at 12 h after treatment with PeBA1 and buffer in tobacco leaves and HSR203J gene expression was induced by PeBA1. (C) PeBA1-induced cell death in infiltrated areas was stained blue (b), buffer treatment areas could not be stained by dye (a). Scale bar = 100 μm.

Fig 4

The minimum concentrations of PeBA1 for HR activity and thermo-stability. (A) Serial dilutions of PeBA1 (20, 10, 5, 1, and 0.5 μM /L) were infiltrated into tobacco leaves, and HR was observed after 24 hours. (B) PeBA1 (50 μl of 20 μM/L) was treated at different temperatures (4, 25, 50, 80, and 100℃) for 15 min, infiltrated into leaves through stoma (using buffer as a control), and photographed at 24 hpi. Infiltrated areas are labelled with circles, and red circles showed typical HR.

Fig 5

Induction of ROS in tobacco leaves by PeBA1. (A) H₂O₂ accumulation in leaves was stained with DAB at 4 h post-inoculation (hpi). (a) PeBA1-infiltrated areas were stained brown compared with (b) buffer treatment as a control. (B) NBT revealed the production of O₂^-, and staining was performed on leaves at 4 hpi. O₂^- accumulation appeared in PeBA1-treated leaves (a), but not in leaves treated with buffer (b). (C) Kinetics of ROS production following elicitation in tobacco cell culture. ROS production was measured by chemiluminescence. PeBA1 rapidly induced ROS formation, with the peak occurring at 4 min. The kinetics are representative of three independent experiments. Error bars represent ± SD of the mean. Values are expressed in arbitrary units (a.u.).

Fig 6

PeBA1-induced phenolic-compound accumulation. Tobacco cell suspension was incubated with PeBA1 or buffer for 108 h. Obvious intense blue fluorescence appeared in the PeBA1-treated cell culture (A) compared with buffer treatment (B). Samples were photographed by confocal microscopy under different lights: fluorescence (left), bright (middle) and overlay (right). Three independent biological replicates were used for the suspension-cultured tobacco cells.

Fig 7

PeBA1-induced resistance in tobacco against Botrytis cinerea. (A) Representative phenotypes of disease caused by B. cinerea in PeBA1- and buffer-induced tobacco leaves. The sizes of the lesions caused by B. cinerea in PeBA1-induced leaves (a) were smaller than in buffer-induced leaves (b) at 96 h post-infiltration. B. Lesion sizes caused by B. cinerea were measured in leaves from PeBA1- or buffer-induced plants. Data presented in (B) are the means ± SD from three independent experiments. The statistical analyses were performed using Student's t test, and asterisks indicate significant differences between PeBA1 and buffer treatment (***, P < 0.001)

Fig 8

PeBA1-induced systemic resistance in tobacco against TMV. Three days after infiltration with PeBA1 or buffer (as a control) on leaves of 6-week-old tobacco plants, systemic leaves were inoculated with TMV. Photographs were taken at 3 days post-inoculation (dpi). (A) Representative phenotypes of disease symptoms caused by TMV infection in tobacco leaves. (A) The numbers and diameters of TMV lesions in systemic leaves of PeBA1-induced plants (b) were significantly fewer and smaller, respectively, than those in control plants (a). (B) Systemic resistance conferred by PeBA1. The numbers of local lesions caused by TMV infection were counted, and lesion sizes were measured at 3 dpi. Values are the means ± SD from three independent experiments. ***P < 0.001 by Student's t-test.

Fig 9

Expression analysis of defence-related genes in tobacco after PeBA1 treatment using buffer as a control. The samples were harvested from systemic leaves at the indicated times, and Q-RT-PCR was performed to show the relative expression levels of the PR-1a, PR1b PR-5, PAL, NPR1, PDF1.2, and COI1 genes. The samples were normalized against Actin, and expression levels are represented as fold changes in relation to the control.