Root-secreted malic acid recruits beneficial soil bacteria

Abstract

Beneficial soil bacteria confer immunity against a wide range of foliar diseases by activating plant defenses, thereby reducing a plant's susceptibility to pathogen attack. Although bacterial signals have been identified that activate these plant defenses, plant metabolites that elicit rhizobacterial responses have not been demonstrated. Here, we provide biochemical evidence that the tricarboxylic acid cycle intermediate L-malic acid (MA) secreted from roots of Arabidopsis (Arabidopsis thaliana) selectively signals and recruits the beneficial rhizobacterium Bacillus subtilis FB17 in a dose-dependent manner. Root secretions of L-MA are induced by the foliar pathogen Pseudomonas syringae pv tomato (Pst DC3000) and elevated levels of L-MA promote binding and biofilm formation of FB17 on Arabidopsis roots. The demonstration that roots selectively secrete L-MA and effectively signal beneficial rhizobacteria establishes a regulatory role of root metabolites in recruitment of beneficial microbes, as well as underscores the breadth and sophistication of plant-microbial interactions.

Figure 1.

B. subtilis strain FB17 root colonization and biofilm formation with pathogen and nonpathogen leaf treatments. The data showed a higher colonization and biofilm formation 5 d posttreatment in response to aerial infection with Pst DC3000 than in untreated (control), water-injected (mock), or nonpathogenic NPS3121-inoculated leaves in representative root colonization experiments by confocal microscopy (A) and CFU quantification (B). The strong green fluorescence along the sides of the roots indicates the FB17 biofilm visualized by staining with SYTO13. Different letters indicate significant difference between the treatments (P < 0.05; ANOVA test; scale = 100 μm). [See online article for color version of this figure.]

Figure 2.

Root exudate composition with pathogen and nonpathogen leaf treatments Greater MA was detected with aerial infection with Pst DC3000 than in untreated (control), water-injected (mock), or nonpathogenic NPS3121-inoculated leaves as observed by HPLC analysis with representative HPLC profiles (arrow indicates MA elution position; A) and MA peak-area quantification (B). Different letters indicate significant difference between the treatments (P < 0.05; ANOVA test).

Figure 3.

MA-specific chemotactic motility of B. subtilis strain FB17 measured by following capillary chemotactic assay. l-MA showed a dose-dependent chemotactic attraction of FB17 (A); structure-dependent chemotactic attraction of FB17 (B); and bacterial species-specific attraction to MA (C). Bacterial strains used in the chemotactic assay included B. subtilis strain FB17, A. tumefaciens strain LBA4404, E. carotovora strain AH2, A. rhizogenes strain Arqua-1, A. brasilense strain Cd, P. fluorescens (Pf01), and P. syringae (Pst DC3000). Data are the average of six replicates from two experiments conducted separately.

Figure 4.

MA transporter mutant (Atalmt1) was ineffective in B. subtilis strain FB17 root recruitment. A, Reduced MA secretion in Atalmt1 with or without Pst DC3000 infection (different letters indicate significant difference between the treatments (P ≤ 0.05; ANOVA test) causes reduced FB17 binding and root colonization, shown with representative confocal images stained with SYTO13 (B). Top two rows = 100 μm; bottom row = 50 μm. C, Quantification of FB17 root binding by CFUs (n = 6). [See online article for color version of this figure.]

Figure 5.

Effect of Pst DC3000 leaf infection on the expression of root AtALMT1. Twenty-day-old Arabidopsis line carrying AtALMT1 promoter∷GUS fusion construct grown on peat pellets was leaf infected with Pst DC3000 and other controls, such as NPS3121, E. coli OP50, and P. aeruginosa PAO1; 12-h-postinfection roots were stained for GUS. The figure shows a significantly higher GUS induction in response Pst DC3000 (on par with the positive control AlCl₃ [4 μm] root treatment) when compared to untreated controls and other bacterial strains such as NPS3121, OP50, and PAO1 (scale = 100 μm).

Figure 6.

A, Effect of Arabidopsis root exudates on the transcription of the yqxM operon in B. subtilis strain Marburg carrying the yqxM-lacZ fusion (NRS1531). Strain NRS1531 was grown in biofilm medium under biofilm formation conditions at 37°C with root exudates (5%) from Pst DC3000-infected plants (triangles), uninfected (squares) plants, and control (diamonds) without root exudates, and β-galactosidase activity was measured at regular intervals and plotted as a function of time. These experiments were repeated on at least three independent occasions and a representative plot is shown. B, Effect of different MA isomers and oxalic acid on the transcription of the yqxM operon in B. subtilis. Strain NRS1531 was grown in biofilm medium under biofilm formation conditions at 37°C with 5 μm MA isomers, l-MA (squares) and d-MA (×), oxalic acid (triangles), control (diamonds) without MA isomers, or oxalic acid, and β-galactosidase activity was measured at regular intervals and plotted as a function of time. These experiments were repeated on at least three independent occasions and a representative plot is shown.

Figure 7.

A schematic depicting the long-distance intraplant signaling to recruit rhizobacterium B. subtilis strain FB17 through secretion of MA postaerial infection by Pst DC3000. [See online article for color version of this figure.]