A Bacillus licheniformis Glycoside Hydrolase 43 Protein Is Recognized as a MAMP

Abstract

Glycoside hydrolases from pathogens have often been reported as inducers of immune responses. However, the roles of glycoside hydrolase from plant-growth-promoting rhizobacteria (PGPR) in the resistance of plants against pathogens is not well studied. In this study, we identified a glycoside hydrolase 43 protein, H1AD43, produced by Bacillus licheniformis BL06 that can trigger defense responses, including cell death. Ion-exchange and size-exclusion chromatography were used for separation, and the amino acid sequence was identified by mass spectrometry. The recombinant protein generated by prokaryotic expression was able to elicit a hypersensitive response (HR) in Nicotiana benthamiana and trigger early defense responses, including reactive oxygen species (ROS) burst, callose accumulation, and the induction of defense genes. In addition, the protein could induce resistance in N. benthamiana, in which it inhibited infection by Phytophthora capsici Leonian and tobacco mosaic virus-green fluorescent protein (TMV-GFP) expression. H1AD43 thus represents a microbe-associated molecular pattern (MAMP) of PGPR that induces plant disease resistance and may provide a new method for the biological control of plant disease.

Keywords: Bacillus licheniformis; glycoside hydrolase 43 family; induced systemic resistance; protein elicitor.

Figure 1

Isolation, purification, and activity verification of the Bacillus licheniformis (BL06) exoprotein. (A) Elution curve of the BL06 exoprotein by HiTrap Capto Q chromatography; (B) Induction of cell death in Nicotiana benthamiana leaves by BL06-1 and BL06-2; (C) The elution curve of BL06-1 by Superdex75 chromatogram; (D) Induction of cell death in N. benthamiana leaves by BL06-1A-1E.

Figure 2

Identification and activity verification of the BL06-1A protein. (A) Mass spectrometry analysis of the BL06-1A protein; (B) Elution curve of H1AD43 in a His-Trap HP column; (C) Induction of cell death in N. benthamiana leaves by H1AD43.

Figure 3

H1AD43 protein property analysis. (A) Evolutionary tree analysis of the GH43 family members from different microbial sources; (B) H1AD43 hydrophilicity and hydrophobicity analysis; (C) H1AD43 structure modeling.

Figure 4

Localization analysis of H1AD43 in N. benthamiana and onion epidermal cells.

Figure 5

Reactive oxygen species burst and callose deposition. (A) H1AD43 triggered an ROS burst in N. benthamiana leaves. (B) H1AD43 induced the accumulation of ROS in N. benthamiana leaves. (C) H1AD43 induced callose deposition.

Figure 6

Expression levels of genes related to defense. (A) Induction of the disease-course-related gene NbPR1 in N. benthamiana after H1AD43 treatment; (B) iInduction of the disease-course-related gene NbPR2 in N. benthamiana after H1AD43 treatment; (C) iInduction of the SA-synthesis-related gene NbPAL in N. benthamiana after H1AD43 treatment; (D) iInduction of the SA-synthesis-related gene NbICS in N. benthamiana treated with H1AD43; (E) iInduction of the PTI gene NbPTI5 in N. benthamiana treated with H1AD43; (F) iInduction of the PTI gene NbCYP71D in N. benthamiana treated with H1AD43; (G) iInduction of the ROS-related gene NbRBOHA in N. benthamiana treated with H1AD43. * represents for p < 0.05 and ** represents for p < 0.01.

Figure 7

H1AD43 induces resistance to Phytophthora capsici and TMV in N. benthamiana. (A) Symptoms of Phytophthora infection; (B) Diameter of diseased spots; (C) Typical symptoms caused by TMV-GFP in tobacco leaves; (D) Number of TMV-GFP diseased spots. * represents for p < 0.05 and *** represents for p < 0.001.