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Epsc Involved in the Encoding of Exopolysaccharides Produced by Bacillus amyloliquefaciens FZB42 Act to Boost the Drought Tolerance of Arabidopsis thaliana

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Epsc Involved in the Encoding of Exopolysaccharides Produced by Bacillus amyloliquefaciens FZB42 Act to Boost the Drought Tolerance of Arabidopsis thaliana

Xiang Lu et al. Int J Mol Sci.

Abstract

Bacillus amyloliquefaciens FZB42 is a plant growth-promoting rhizobacteria that stimulates plant growth, and enhances resistance to pathogens and tolerance of salt stress. Instead, the mechanistic basis of drought tolerance in Arabidopsis thaliana induced by FZB42 remains unexplored. Here, we constructed an exopolysaccharide-deficient mutant epsC and determined the role of epsC in FZB42-induced drought tolerance in A. thaliana. Results showed that FZB42 significantly enhanced growth and drought tolerance of Arabidopsis by increasing the survival rate, fresh and dry shoot weights, primary root length, root dry weight, lateral root number, and total lateral root length. Coordinated changes were also observed in cellular defense responses, including elevated concentrations of proline and activities of superoxide dismutase and peroxidase, decreased concentrations of malondialdehyde, and accumulation of hydrogen peroxide in plants treated with FZB42. The relative expression levels of drought defense-related marker genes, such as RD29A, RD17, ERD1, and LEA14, were also increased in the leaves of FZB42-treated plants. In addition, FZB42 induced the drought tolerance in Arabidopsis by the action of both ethylene and jasmonate, but not abscisic acid. However, plants inoculated with mutant strain epsC were less able to resist drought stress with respect to each of these parameters, indicating that epsC are required for the full benefit of FZB42 inoculation to be gained. Moreover, the mutant strain was less capable of supporting the formation of a biofilm and of colonizing the A. thaliana root. Therefore, epsC is an important factor that allows FZB42 to colonize the roots and induce systemic drought tolerance in Arabidopsis.

Keywords: Bacillus amyloliquefaciens FZB42; biofilm; drought stress; epsC; exopolysaccharides; phytohormone.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The effect of inoculation with either wild type FZB42 or the epsC mutant on the performance of A. thaliana seedlings. Appearance of the plants. Scale bar, 2 cm (A), seedling survival rate under drought stress (B), shoot fresh weight (C), shoot dry weight (D), root dry weight (E), primary root length (F), lateral root number (G), total lateral root length (H). Values in (B) through (H) are shown as means, with the whiskers representing the standard error (SE, n = 18). Different letters above each column indicate statistically significant (p < 0.05) differences in mean performance.
Figure 2
Figure 2
The biochemical response of the A. thaliana leaf to drought stress. The concentration of proline and malondialdehyde (MDA), and the activity of superoxide dismutase (SOD) and peroxidase (POD) (A), 3,3-diaminobenzidine staining revealing the accumulation of hydrogen peroxide in well-watered and droughted plants inoculated with either wild type FZB42 or the epsC mutant. Scale bar, 1 cm (B), 3,3-diaminobenzidine staining revealing the accumulation of hydrogen peroxide in wild type Arabidopsis leaves treated with H2O or 20 mM H2O2. Scale bars, 1 mm (C). Values in (A) are shown as means, with the whiskers representing the standard error (SE, n = 18). Different letters above each column indicate statistically significant (p < 0.05) differences in mean performance.
Figure 3
Figure 3
The physiological response of the A. thaliana leaf to drought stress. Appearance of stoma. Scale bar, 2.5 µm (A), and stomatal aperture in detached leaves of plants inoculated with either wild type FZB42 or the epsC mutant (B). Values are shown as means, with the whiskers representing the standard error (SE, n = 18). Different letters above each column indicate statistically significant (p < 0.05) differences in mean performance.
Figure 4
Figure 4
The relative expression levels of drought-responsive marker genes in the leaves of A. thaliana. A qRT-PCR assay was used to estimate the expression levels of the stress response-associated marker genes, RD29A, RD17, ERD1, and LEA14, in drought plants inoculated with either wild type FZB42 or the epsC mutant. Values are shown as means, with the whiskers representing the standard error (SE, n = 18). Different letters above each column indicate statistically significant (p < 0.05) differences in mean performance.
Figure 5
Figure 5
The effect of inoculation with either wild type FZB42 or the epsC mutant on the accumulation of dry matter by well-watered and drought seedlings of Arabidopsis mutants ein2-1 (ethylene insensitive), eto1-1 (ethylene over-producer), jar1-1 (jasmonate insensitive), abi4-102 (ABA insensitive), ga1 (gibberellin-responsive male-sterile dwarf), npr1-1 (salicylic acid insensitive), and the npr1-1/jar1-1 double mutant. Dry weight of Arabidopsis mutants under well-watered condition (A), dry weight of Arabidopsis mutants under drought condition (B). Values are shown as means, with the whiskers representing the standard error (SE, n = 18). Different letters above each column indicate statistically significant (p < 0.05) differences in mean performance.
Figure 6
Figure 6
Biofilm formation by wild type FZB42 and the epsC mutant. The upper panel illustrates the formation of a pellicle by cells cultured at 30 °C for three days on LBGM medium (Lysogeny broth supplemented with 1% (v/v) glycerol and 0.1 mM MnSO4); the lower panel illustrates colony morphology (A), quantitative spectrophotometric assay of biofilms stained with crystal violet (B), confocal laser scanning microscopy analysis of root colonization. Scale bar, 100 µm (C), adherence capacities of wild type and epsC mutant to the A. thaliana root (D). Values in (B) and (D) are shown as means, with the whiskers representing the standard error (SE, n = 12). Different letters above each column indicate statistically significant (p < 0.05) differences in mean performance.
Figure 7
Figure 7
High performance anion exchange chromatography analysis of the monosaccharide composition of exopolysaccharides formed by wild type FZB42 cells. Peaks 8, 9, and 10 correspond to, respectively, galactose, glucose, and mannose. Red line represented the base line. Additional signals occurring between 28 and 32 min were system peaks.
Figure 8
Figure 8
Construction of epsC double-crossover deletion mutants.

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