Purification, Identification And Characterization Of A Novel Elicitor PeBA1 From Bacillus Amyloliquefaciens NC6

Bacillus amyloliquefaciens is a very important kind of plant root-colonizing PGPR in biocontrol for plant diseases. Bacillus spp. can be used as a biocontrol agent via competition, antibiosis, induced systemic resistance, and growth-promoting. In our research, we report the purification, identification and characterization of a novel protein elicitor PeBA1 from the B. amyloliquefaciens NC6, which can induce a typical HR in tobacco leaves and activate defence-related early events. And further study, we find that PeBA1 enhances tobacco disease resistance to TMV and Botrytis cinerea by triggering defence-related gene up-regulation and accumulation of antimicrobial compounds. These results indicate that our research helps to elucidate the mechanisms of B. amyloliquefaciens NC6-induced systemic resistance in tobacco.1. Isolated and purification of protein elicitor and mass spectrometryWe isolated and purified a novel protein elicitor from B. amyloliquefaciens NC6 by precipitated with ammonium sulfate, ion-exchange chromatography and native-PAGE extraction. And the protein induced HR in tobacco leaves. The amino acid sequence was identified by mass spectrometry and named PeBA1(Protein elicitor from Bacillus amyloliquefaciens 1).2. The expression and purification of PeBA1We designed a pair of primers to clone PeBA1 from genomic DNA of B. amyloliquefaciens NC6. Then PeBA1 was cloned to E1 plasmid and transformed into E. coli BL21(DE3) cells. Further purification was executed by affinity chromatography with Ni column and we obtained soluble recombinant PeBA1. Recombinant PeBA1 induced HR activity in tobacco leaves and exhibited a single band with an apparent molecular mass of approximately 34 kDa on an SDS-PAGE gel, which was in agreement with the calculated size of the recombinant protein,3. Characterization of recombinant PeBA1 and triggered defense responses in tobaccoWe found that the minimum concentration of recombinant PeBA1 for HR activity was 5μM and the recombinant PeBA1 retained its activity to induced HR after treatment with 4、25、50、80、100℃ for 15 min. Recombinant Pe BA1 activated defence-related early events, such as an ROS(H2O2 and O2-) burst. And Recombinant PeBA1 could also induce the accumulation of Phenolic compounds to enhance mechanical strength of the cell wall to protect against infection with pathogenic bacteria or fungi.4. PeBA1 induced systemic resistance in tobaccoQuantitative real-time PCR(Q-RT-PCR) results indicated that several plant defence genes, including the salicylic acid(SA)-responsive PR1 a, PR1 b, 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.