Isolation and identification of a novel protein elicitor from a Bacillus subtilis strain BU412

Abstract

Here, we report a novel protein elicitor from Bacillus subtilis BU412 which could cause hypersensitive response (HR) and systemic acquired resistance (SAR) in tobacco. The purification was executed by ion-exchange and size exclusion chromatography. The target band on SDS-PAGE was analyzed by mass spectrometry, and the peptide mass fingerprinting matched an uncharacterized protein (WP_017418614.1), which was then named AMEP412. AMEP412 could cause a clearly defined HR necrosis in tobacco leaves, which was less affected by thermal treatment. The sub-cellular localization assay revealed that AMEP412 localized on the cell surface. This protein could also trigger early defense events such as the generation of reactive oxygen species (H₂O₂ and O₂^-) and the induction of defense enzymes, including superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL). Moreover, AMEP412 could stimulate plant systemic resistance against Pseudomonas syringae pv. tomato DC3000.

Keywords: Bacillus subtilis; Hypersensitive response; Induced systemic resistance; Protein elicitor; Reactive oxygen species.

Fig. 1

The purification of the new elicitor protein. a The anion exchange chromatography map. P1-P4 were peaks eluted with a linear gradient of NaCl. b The superdex chromatography map. F1 and F2 were peaks eluted with 20 mM Tris–HCl. Inset, typical chromatogram from a function test of Superdex 75 10/300 GL (Amersham Biosciences). c The SDS-PAGE detection of the target protein. M: low molecular weight standards, 1: the purified protein sample of the peak F2, B1: the target protein band

Fig. 2

AMEP412 induced HR in tobacco leaves. a HR lesion caused by AMEP412 in tobacco leaves. b Trypan blue staining of the HR areas infiltrated by AMEP412. Buffer treatment areas could not be stained by dye. AMEP412 induced cell death in infiltrated areas was stained blue. Scale bar = 50 μm

Fig. 3

The characterization of AMEP412 for its HR activity. a The minimum concentration of AMEP412 for its HR activity. Serial dilutions of AMEP412 (2.4, 2.0, 1.6, 1.2, 0.8 and 0.4 mg mL⁻¹) were infiltrated into tobacco leaves, and HR was observed after 24 h. b The thermal stability of AMEP412 for its HR activity. AMEP412 was treated at different temperatures (25, 40, 60, 80 and 100 °C) for 5 min, infiltrated into leaves and photographed at 24 h

Fig. 4

Sub-cellular localization of AMEP412 in tobacco cells

Fig. 5

Induction of ROS in tobacco leaves by AMEP412. a DAB showed the production of H₂O₂. The leaves were stained at different post treatment hours. H₂O₂ accumulation appeared in AMEP412 treated leaves. b NBT revealed the production of O₂⁻, and staining was performed on leaves at the same treatment time. O₂⁻ accumulation appeared in AMEP412 treated leaves

Fig. 6

Kinetics of SOD, POD, PPO and PAL activity after AMEP412 treatment. The activities were measured 0–72 h after AMEP412 treatment. The values are the mean ± SD of quintuplicate samples

Fig. 7

AMEP412 induced systemic resistance in tobacco against Pst DC3000