Identification And Gene Characterization Of A Novel Elicitor PeBL1 From Brevibacillus Laterosporus Strain A60

Application of biocontrol agents for controlling plant diseases have become the focus of attention. Brevibacillus laterosporus strain A60, as a biocontrol agent, can induce plant systemic resistance against infection of plant diseases. In this paper, a protein elicitor, PeBL1, from the culture filtrate of Brevibacillus laterosporus strain A60 was isolated and purified. PeBL1 can induce defense responses and systemic resistance in Nicotiana benthamiana. The elicitor-treated tobaccos exhibit strong resistance to the infection of tobacco mosaic virus-green fluorescent protein (TMV-GFP) and Pseudomonas syringae pv. tabaci compared to control tobacco. The PeBLl gene was cloned and expressed in Escherichia coli. The recombinant protein exhibits the same biological activity as the endogenous protein. Real-time quantitative-PCR analysis showed that resistance signaling pathway in N. benthamiana induced by PeBLl. This research helps to elucidate the mechanisms of N. benthamiana systemic resistance triggered by PeBLl and provides a novel strategy for using strain A60 to control plant disease.1. Through a purification process consisting of ion-exchange chromatography and high-performance liquid chromatography (HPLC), we isolated and purified a protein elicitor, PeBLl (Protein elicitor from Brevibacillus laterosporus 1), from the culture filtrate of Brevibacillus laterosporus strain A60. PeBLl can induce the typical HR and didn’t show pH and thermal stability. PeBLl was purified to homogeneity without any carbohydrate moieties.2. PeBLl was identified by electrospray ionization quadrupole time of flight tandem mass spectrometry (ESI-Q-TOF-MS/MS). We obtained the best-matching hypothetical protein from B. laterosporus. PeBL1 contains 116 amino acids with a theoretical molecular mass of 12833 Da. We considered the hypothetical protein was PeBL1 and cloned the PeBL1 gene with 351bp. The PeBLl sequence was cloned into the pET30-TEV/LIC vector. We got the pure recombinant protein His-PeBLl which could also induce the HR in tobacco leaves.3. The PeBLl-treated tobacco can elicit the defense responses, including that ROS burst, extracellular medium alkalization, phenolic-compound and lingin deposition, and exhibit strong resistance to the infection of TMV-GFP and Pseudomonas syringae pv. tabaci. The greatest reductions in number and size of lesion were 42.91% and 43.23% respectively at 4 days post inoculation. Also the population of Pseudomonas syringae pv. tabaci had decreased by 30% at 4 days post inoculation.4. Several defense-related genes were upregulated to varying degrees by PeBLl, indicating that expression of defense-related genes may be a mechanism of induced plant resistance. The up-expression of NPR1, PR-1α, PR-5 and PDF1.2 genes demonstrated that the PeBLl induces systemic resistance using a complex signaling network, which most likely includes SA-and JA/ET-dependent pathways.