Geng C, Nie X, Tang Z, Zhang Y, Lin J, Sun M, Peng D.

Sci Rep. 2016 Apr 27;6:25012. doi: 10.1038/srep25012.

Abstract

Plant-parasitic nematodes (PPNs) cause serious harm to agricultural production. Bacillus firmus shows excellent control of PPNs and has been produced as a commercial nematicide. However, its nematicidal factors and mechanisms are still unknown. In this study, we showed that B. firmus strain DS-1 has high toxicity against Meloidogyne incognita and soybean cyst nematode. We sequenced the whole genome of DS-1 and identified multiple potential virulence factors. We then focused on a peptidase S8 superfamily protein called Sep1 and demonstrated that it had toxicity against the nematodes Caenorhabditis elegans and M. incognita. The Sep1 protein exhibited serine protease activity and degraded the intestinal tissues of nematodes. Thus, the Sep1 protease of B. firmus is a novel biocontrol factor with activity against a root-knot nematode. We then used C. elegans as a model to elucidate the nematicidal mechanism of Sep1, and the results showed that Sep1 could degrade multiple intestinal and cuticle-associated proteins and destroyed host physical barriers. The knowledge gained in our study will lead to a better understanding of the mechanisms of B. firmus against PPNs and will aid in the development of novel bio-agents with increased efficacy for controlling PPNs.

See: https://www.ncbi.nlm.nih.gov/pubmed/27118554

Figure 3: Sep1 protein shows toxicity against nematodes.

(A) The transcript levels of sep1 mRNA in Bacillus firmus DS-1 at different phases of bacterial growth. (B) Growth assay of Sep1 against C. elegans. All nematodes are shown at the same magnification (scan bar 100 μm). (C) The growth inhibition curve for the growth assay. The highest dose of (100%) represents Sep1-expressing E. coli BL21 (OD₆₀₀ = 0.6); other doses were diluted with E. coli BL21 harboring an empty vector (Control). The inhibition percentage was the ratio of average worm size treated with Sep1 relative to the average size of control animals. (D) The mortality assays of Sep1 against C. elegans. The lethal activity of E. coli-expressed Sep1 (OD₆₀₀ = 0.6) was tested with N2 L4 nematodes at different times. The same amount of E. coli BL21 harboring an empty vector was used as a control (CK). Each point represents the average result from 20–30 animals. (E) Mortality assay of Sep1 proteins against M. incognita J2. The 1 μg/ml RES solution-treated animals were used as controls (CK). Each point represents the average result from 50–60 animals. All the data are shown as the mean ± SD (n = 3), statistical comparisons between the values of detect samples and CKs were performed using a paired t-test, and significant differences were determined according to a threshold of *p < 0.05; **p < 0.01, and ***p < 0.001.