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. 2019 Apr 18;85(9):e00178-19.
doi: 10.1128/AEM.00178-19. Print 2019 May 1.

Stress-Responsive Alternative Sigma Factor SigB Plays a Positive Role in the Antifungal Proficiency of Bacillus subtilis

M Bartolini  1 , S Cogliati  1 , D Vileta  1 , C Bauman  1 , W Ramirez  1 , R Grau  2
Affiliations

Stress-Responsive Alternative Sigma Factor SigB Plays a Positive Role in the Antifungal Proficiency of Bacillus subtilis

M Bartolini et al. Appl Environ Microbiol. .

Abstract

Different Bacillus species with PGPR (plant growth-promoting rhizobacterium) activity produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how these PGPRs recognize phytopathogens and exhibit the antifungal response. Here, we report the antagonistic interaction between Bacillus subtilis and the phytopathogenic fungus Fusarium verticillioides We demonstrate that this bacterial-fungal interaction triggers the induction of the SigB transcription factor, the master regulator of B. subtilis stress adaptation. Dual-growth experiments performed with live or dead mycelia or culture supernatants of F. verticillioides showed that SigB was activated and required for the biocontrol of fungal growth. Mutations in the different regulatory pathways of SigB activation in the isogenic background revealed that only the energy-related RsbP-dependent arm of SigB activation was responsible for specific fungal detection and triggering the antagonistic response. The activation of SigB increased the expression of the operon responsible for the production of the antimicrobial cyclic lipopeptide surfactin (the srfA operon). SigB-deficient B. subtilis cultures produced decreased amounts of surfactin, and B. subtilis cultures defective in surfactin production (ΔsrfA) were unable to control the growth of F. verticillioidesIn vivo experiments of seed germination efficiency and early plant growth inhibition in the presence of F. verticillioides confirmed the physiological importance of SigB activity for plant bioprotection.IMPORTANCE Biological control using beneficial bacteria (PGPRs) represents an attractive and environment-friendly alternative to pesticides for controlling plant diseases. Different PGPR Bacillus species produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how PGPRs recognize phytopathogens and process the antifungal response. Here, we report how B. subtilis triggers the induction of the stress-responsive sigma B transcription factor and the synthesis of the lipopeptide surfactin to fight the phytopathogen. Our findings show the participation of the stress-responsive regulon of PGPR Bacillus in the detection and biocontrol of a phytopathogenic fungus of agronomic impact.

Keywords: Bacillus subtilis; bicontrol; fungi; sigma B; stress; surfactin.

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Figures

FIG 1
FIG 1
Antagonistic response of B. subtilis confronted with F. verticillioides. (A and B) The coculture of a B. subtilis NCIB3610 isogenic strain harboring a ctc-lacZ fusion as a reporter of SigB activity (strain DG555) (Table 1) with F. verticillioides allowed observation of the antagonistic fungus-bacterium interaction. The pattern of induction of SigB is evidenced by the development of blue color (derived from expression of the ctc-lacZ fusion) inside the bacterial colony. The areas represented by the rectangles correspond to the colony areas that were used to quantify the level of SigB-directed β-galactosidase activity (see the text for details). (C) Pattern of SigB expression when the DG555 strain was developed in the absence of F. verticillioides. For panels A to C, cells were grown on PDA plates supplemented with X-Gal (60 µg/ml) for 96 h at 28°C. (D and E) Planktonic growth and sporulation proficiency of the wild-type (wt) strain NCIB3610 and its isogenic SigB-deficient derivative (ΔsigB; strain DG559) (Table 1) in the absence and presence of live F. verticillioides (see Materials and Methods for details). Typical results from five independent experiments performed in duplicate are shown for panels A to E.
FIG 2
FIG 2
F. verticillioides induces the stress-responsive SigB regulon. β-Galactosidase activity of LB cultures of the wild-type strain DG555 in response to different amounts of live (A), dead (B), or cell-free supernatant (C) of F. verticillioides. β-Galactosidase values are expressed in MU ± standard errors of the means (SEM), and time zero corresponds to the moment that the bacterial cultures reached the mid-logarithmic phase of growth (OD600 of 0.5) and fungal addition (see Materials and Methods for details). A typical output from three independent experiments performed in parallel is shown.
FIG 3
FIG 3
B. subtilis recognizes F. verticillioides via the energy-dependent pathway of the SigB regulatory cascade. (A) A cartoon summarizing the three known pathways of SigB activation, one of which is likely responsible for sensing the presence of the fungus (see the text for details). (B to F) β-Galactosidase activity of NCIB3610 isogenic strains harboring the ctc-lacZ fusion in wild-type background (strain DG555) (E and F) or affected in the different pathways of SigB activation: ΔrsbU (strain DG556) (B), ΔrsbP (strain DG557) (C), and ΔrsbUP (strain DG558) (D and F). Each bacterial culture was grown in LB broth with shaking at 28°C (B to D), 37°C (E), or the indicated temperatures (F) until the mid-logarithmic phase, at which time (time zero) the culture was divided into two subcultures and F. verticillioides was added to one of them (final fungal concentration, 1%). The incubation was continued as shown in the figure, and aliquots for the determination of β-galactosidase activity were taken at the indicated times and processed. For the experiment shown in panel F, β-galactosidase activity was determined 40 min after time zero. For panels B to F, a typical output from three independent experiments performed in parallel is shown.
FIG 4
FIG 4
Role of SigB in the in vitro antifungal activity of B. subtilis. (A and B) F. verticillioides (A) and the wild-type B. subtilis strain NCIB3610 and its isogenic ΔsigB strain (DG559) (B) were grown on PDA plates at 28°C as indicated in Materials and Methods. (C) Four-day growth of F. verticillioides inoculated in the middle of a PDA plate without supplementation (left plate) or supplemented with 10% culture supernatant from NCIB3610 (wild type) or DG599 (ΔsigB) B. subtilis cultures. (D) Growth of F. verticillioides under axenic conditions or cocultured with the wild-type strain NCIB3610 or the isogenic SigB-deficient derivative (ΔsigB). Cultures were developed in LB broth with shaking at 28°C, and fungal quantification (CFU ml−1 ± SEM) at different times of growth was carried out as described in Materials and Methods. The results from five independent experiments performed in duplicate are shown in panel D, and panels A through C show representative results.
FIG 5
FIG 5
Surfactin has an essential role in the in vitro antifungal activity of B. subtilis. (A and B) Absence of antifungal activity of an NCIB3610 isogenic strain deficient in surfactin production (ΔsrfA, strain DG560) (Table 1). (C) Growth of F. verticillioides in the absence or presence of the surfactin-deficient derivative (ΔsrfA) DG560 in LB broth with shaking at 28°C as described in Materials and Methods. The results of five representative experiments are shown.
FIG 6
FIG 6
SigB-dependent surfactin production. β-Galactosidase activity of NCIB3610 isogenic strains, proficient and deficient in SigB activity, harboring srf-lacZ::amyE as a reporter of surfactin production (strains DG561 [wt], DG562 [ΔsigB], DG563 [ΔrsbP], and DG564 [ΔrsbU]). Each bacterial culture (with or without 1% fungal addition) was grown in LB broth with shaking at 28°C and processed as indicated in Materials and Methods.
FIG 7
FIG 7
Biocontrol proficiency of PGPR B. subtilis: in vivo roles of SigB and surfactin. (A and B) Germination efficiency (A) and plant growth (root length) (B) of Zea mays infected with F. verticillioides in the absence or presence of B. subtilis (see Materials and Methods for details). A typical output of three independent experiments is shown. (C) A cartoon summarizing the beneficial interactions between surfactin-producing PGPR B. subtilis cells and plants to resist phytopathogenic fungi. For simplicity, the stimulatory effect of plant polysaccharides on biofilm formation and surfactin synthesis is not indicated (see the text for details).
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