Sucrose triggers a novel signaling cascade promoting Bacillus subtilis rhizosphere colonization

Abstract

Beneficial rhizobacteria promote plant growth and protect plants against phytopathogens. Effective colonization on plant roots is critical for the rhizobacteria to exert beneficial activities. How bacteria migrate swiftly in the soil of semisolid or solid nature remains unclear. Here we report that sucrose, a disaccharide ubiquitously deployed by photosynthetic plants for fixed carbon transport and storage, and abundantly secreted from plant roots, promotes solid surface motility (SSM) and root colonization by Bacillus subtilis through a previously uncharacterized mechanism. Sucrose induces robust SSM by triggering a signaling cascade, first through extracellular synthesis of polymeric levan, which in turn stimulates strong production of surfactin and hyper-flagellation of the cells. B. subtilis poorly colonizes the roots of Arabidopsis thaliana mutants deficient in root-exudation of sucrose, while exogenously added sucrose selectively shapes the rhizomicrobiome associated with the tomato plant roots, promoting specifically bacilli and pseudomonad. We propose that sucrose activates a signaling cascade to trigger SSM and promote rhizosphere colonization by B. subtilis. Our findings also suggest a practicable approach to boost prevalence of beneficial Bacillus species in plant protection.

Fig. 1. Sucrose promotes root colonization and solid surface motility (SSM) by B. subtilis.

A LSCM images of 18-day-old tomato roots observed 72 h after inoculation with a B. subtilis 3610 derivative constitutively expressing mKate2 and treated with different sugars (Fru fructose, Glc glucose, Mal maltose, and Suc sucrose). For treatments of sugars, 5 ml cell suspension supplemented with 0.5% (w/v) sugar was applied to the roots. Water indicates the same volume of water replacing any sugar solution. CK indicates no addition of bacterial cells and sugars. Shown pictures are representatives of at least 20 independent root samples (scale bars: 50 μm). B The influence of different sugar supplementations on the colonization of 3610 cells on tomato roots was determined by counting colony forming unit (CFU) per mm root length. Error bars represent standard deviations. * indicates p value < 0.05; ** indicates p value < 0.01; NS no statistical difference. C The concentration of supplementary sucrose positively correlated with the robustness of solid-surface movement (SSM) by 3610. The microscopic images of outer-edge cells stained with flagella-specific dye, on solid LB plate with or without sucrose (5 g/L). Pictures are representatives of at least five independent samples (scale bars: 3 μm). D The increasing concentrations of agar negatively influenced the robustness of sucrose-induced SSM. E Colony expanding rate of 3610 on solid LB plate with or without supplementary sucrose (5 g/L) was determined by measuring the diameter of the colony periodically. Assays were done in triplicate. Error bars represent standard deviations. F Addition of sucrose (5 g/L) also triggered SSM by other Bacillus strains on solid LB plates. All Petra dishes shown here have a diameter of 10 cm.

Fig. 2. Surfactin mediates sucrose-induced SSM by B. subtilis.

A The effect of various sugars on surfactin yield (µg/cm²) by B. subtilis 3610 cells was assayed on solid LB agar plates (1.5% agar, w/v). Samples were collected from the plates supplemented with different sugars, all at the concentration of 5 g/L. Surfactin was extracted, and the amount of surfactin was determined by HPLC as described in the method. The error bars represent standard deviations from triplicate assays. * indicates p value < 0.05; ** indicates p value < 0.01; NS no statistical difference. B Microscopy images of cells harboring the promoter fusion (P_srfAA-gfp, CY106) collected from the edge of the colonies after 4 h of inoculation on solid LB plates with or without 5 g/L sucrose (scale bars: 5 μm). C Quantification of fluorescence intensity of the cells expressing P_srfAA-gfp from above. The quantification for each sample is based on roughly 200 cells by using ImageJ. Solid lines in the middle indicates the mean value (artificial units, AU) of the fluorescence intensity. Upper and lower dotted lines indicate the 75% and 25% quartile, respectively. D Assays of β-galactosidase activities of cells bearing the P_srfAA-lacZ promoter fusion (KG203). Cells were similarly collected from the edge of the colonies after 4 h inoculation from solid LB agar plates without sugar addition, with the addition of 5 g/L sucrose or glucose. Assays were done in biological triplicates. Error bars represent standard deviations. ** indicates p value < 0.01; NS, no statistical difference. E The srfAA mutants of three B. subtilis strains (9407, NCD-2, and 3610) lost SSM on solid LB with 5 g/L sucrose plates when compared to their wild-type counterparts. Images are representatives of at least 3 independent assays. F Pure surfactin, but not sucrose, rescues the phenotype of SSM and hyper-flagellation in a srfAA mutant of 3610 (Tm01). Solutions (in a volume of 100 μL) containing 180 μg surfactin were filled into an Oxford-cup, which is 1 cm away from the inoculating spot with the B. subtilis cells. All Petra dishes shown here have a diameter of 10 cm.

Fig. 3. Levan induces SSM in B. subtilis and other soil bacteria.

A A schematic diagram of sucrose metabolism and the signal relay triggering SSM in B. subtilis. On the left of the diagram, it indicates that the extracellular sucrose is imported into cells by the SacP transporter, and then hydrolyzed into glucose-6-P and fructose by the SacA hydrolase. On the top, it illustrates that sucrose is metabolized extracellularly. The levansucrase SacB uses sucrose to synthesize polymeric fructoses (levan), and glucose. When needed, levan can be degraded into levanoligosaccharides by LevB, and into monomeric fructoses by SacC, which is then imported into cells by the transporter composed of LevDEFG. Levan likely indirectly actives the srfAA-AD operon, whose product (surfactin) triggers SSM in B. subtilis. B SSM by B. subtilis 3610 and various mutants on solid LB plates supplemented with sucrose (5 g/L), levan (0.2 g/L), or levanbiose (0.2 g/L). Pictures are representatives of at least three independent assays. C Pure levan, when supplied at 0.2 g/L, induces SSM and hyper-flagellation in B. subtilis 3610. D The structural analogues of levan (inulin, FOS, and dextran) cannot induce SSM by B. subtilis 3610 when provided at 0.2 g/L. E Levan (0.2 g/L) induces SSM by some soil bacteria (Serratia marcescens T4-3, Pectobacterium carotovorum subsp. carotovorum Z3-3, Xanthomonas oryzae pv. oryzae PXO99F, Pseudomonas protegens pf-5, and P. fluorescens 2p24). All pictures are representatives of at least three independent assays. All Petra dishes shown here have a diameter of 10 cm.

Fig. 4. Sucrose promotes competitive root colonization by B. subtilis.

A LSCM images of root colonization by WT (3610) and the mutants (ΔsacA and ΔsacB) treated without or with sucrose (+S). B The difference on the tomato root colonization between the WT and the two mutants was determined by counting colony forming unit (CFU) per mm root length. Error bars represent standard deviations. ** indicates p value < 0.01; * indicates p value < 0.05. C The colonization competition between 905 and 3610 (including its derivates) on tomato roots was determined by counting CFU per mm root length. * indicates p value < 0.05; ** indicates p value < 0.01; NS, no statistical difference. D The pictures of seedlings of wild-type A. thaliana (Col-0) and its derivatives impaired in sucrose transport [ΔAtSUC2 (At1g22710, SALK_0038124), ΔAtSTP1 (At1g11260, SALK_048848c), and ΔAtSUC3 (At2g02860, SALK_077723)]. Weakened developmental effects were observed in some of the mutants. Pictures are representative of at least 20 independent plants (Scale bars: 1.4 cm). E LSCM pictures of 15-day-old roots of wild-type A. thaliana and the mutants. The roots were observed 72 h after inoculation with strain 3610 constitutively expressing mKate2. Pictures are representative of at least 20 independent roots (Scale bars: 50 μm). F The influence of different sugar transporters on the colonization of 3610 on the A. thaliana roots was determined using colony forming unit (CFU) per mm root length by plate recovery counting. The letters above the columns indicate statistically significant differences of different groups based on Student’s t test (p < 0.01).

Fig. 5. Sucrose selectively shapes rhizomicrobiome and enhances disease control by B. subtilis.

A The relative abundance of 18 top genus groups. Community barplot analysis of bacterial taxonomic groups (genus level) in the tomato root rhizosphere with B. subtilis 3610 or ΔsacB, and with or without the addition of sucrose. Different treatments are marked as: A (WT), AS (WT + sucrose), B (ΔsacB), BS (ΔsacB + sucrose), C (-sucrose), and CS (+sucrose). The numbers 1–3 represent three repetitions. The 18 different colors represent different bacterial genus. The Bacillus and Pseudomonas groups are highlighted by arrows. B The ratio of Bacillus and Pseudomonas species in the microbiome samples based on the relative value of averaged 16S rRNA gene copies per sample (16S rRNA copies/g soil) in the rhizomicrobiome samples with B. subtilis 3610 or ΔsacB mutant in sucrose and no sucrose conditions. Note: A B. subtilis 3610, AS B. subtilis 3610 plus sucrose, B ΔsacB mutant, BS ΔsacB mutant plus sucrose, C no 3610 and sucrose, and CS with only sucrose. Values are given as means of three independent biological replicates and the bars represent the standard error. The letters above the columns indicate statistically significant differences based on the 16S rRNA gene copies of Bacillus or Pseudomonas per sample and using the Student’s t test (p < 0.01). C Combination of sucrose and the B. subtilis improved the biological control efficiency against the soil-borne disease (fusarium wilt) and the resistance to the air-borne disease (gray mold) in tomato. ** indicates p value < 0.01; NS no statistical difference. The error bars represent standard deviations from triplicate assays.