A prominent role of the flagellin receptor FLAGELLIN-SENSING2 in mediating stomatal response to Pseudomonas syringae pv tomato DC3000 in Arabidopsis

Abstract

The FLAGELLIN-SENSING2 (FLS2) receptor kinase recognizes bacterial flagellin and initiates a battery of downstream defense responses to reduce bacterial invasion through stomata in the epidermis and bacterial multiplication in the apoplast of infected plants. Recent studies have shown that during Pseudomonas syringae pv tomato (Pst) DC3000 infection of Arabidopsis (Arabidopsis thaliana), FLS2-mediated immunity is actively suppressed by effector proteins (such as AvrPto and AvrPtoB) secreted through the bacterial type III secretion system (T3SS). We provide evidence here that T3SS effector-based suppression does not appear to be sufficient to overcome FLS2-based immunity during Pst DC3000 infection, but that the phytotoxin coronatine (COR) produced by Pst DC3000 also plays a critical role. COR-deficient mutants of Pst DC3000 are severely reduced in virulence when inoculated onto the leaf surface of wild-type Columbia-0 plants, but this defect was rescued almost fully in fls2 mutant plants. Although bacteria are thought to carry multiple microbe-associated molecular patterns, stomata of fls2 plants are completely unresponsive to COR-deficient mutant Pst DC3000 bacteria. The responses of fls2 plants were similar to those of the Arabidopsis G-protein alpha subunit1-3 mutant, which is defective in abscisic acid-regulated stomatal closure, but were distinct from those of the Arabidopsis non-expressor of PR genes1 mutant, which is defective in salicylic acid-dependent stomatal closure and apoplast defense. Epistasis analyses show that salicylic acid signaling acts upstream of abscisic acid signaling in bacterium-triggered stomatal closure. Taken together, these results suggest a particularly important role of FLS2-mediated resistance to COR-deficient mutant Pst DC3000 bacteria, and nonredundant roles of COR and T3SS effector proteins in the suppression of FLS2-mediated resistance in the Arabidopsis-Pst DC3000 interaction.

Figure 1.

fls2 and Ws-0 plants exhibit enhanced susceptibility to COR-deficient mutant bacteria. Leaf appearances (A and C) and bacterial populations (B and D) 3 d after surface inoculation with Pst DC3118 at 1E + 8 CFU/mL. Results are displayed as means of four different leaves from four different plants, with ses indicated. Statistical differences are detected with ANOVA (P = 0.0001) followed by Turkey's multiple comparison test (showing comparisons to Col-0; ***, P < 0.001) for B, and with two-tailed t test (**, P < 0.01) for D.

Figure 2.

Stomatal closure responses to COR-deficient or wild-type bacteria. Stomatal apertures from leaf peels of Col-0, fls2, efr-1, and fls2 efr-1 plants treated with water or Pst DC3118 at 1E + 8 CFU/mL (A), Col-0 and Ws-0 plants treated with water or Pst DC3118 at 1E + 8 CFU/mL (B), and Col-0 and fls2 plants treated with water or Pst DC3000 at 1E + 8 CFU/mL (C). Results are displayed as means of 30 to 60 stomata, with ses indicated. Statistical differences between water and bacterial treatment are detected with two-tailed t test (***, P < 0.001). In all our experiments, to preserve the stomatal aperture status in plants used for both stomatal and bacterial pathogenesis assays, we did not further treat leaf peels in any stomatal opening buffer.

Figure 3.

Entry of Pst DC3118-GFP into Col-0 and fls2 leaves detected by confocal microscopy. A, A Col-0 leaf with sampling areas indicated by squares. B, Numbers of bacteria observed, showing means from six microscopic views (0.1 mm² each) with ses. Statistical difference is detected with a two-tailed t test (**, P < 0.01). C and D, Side views of three-dimensional models reconstructed from overlaid z-sections of GFP channel in Col-0 and fls2 leaves, respectively. The dimensions of the leaf section (in μm) of axis (x, z) are labeled. Please note that GFP-labeled bacteria (green dots) are inside the leaves.

Figure 4.

Susceptibility of plants to Pst DC3000 or Pst DC3118 when inoculated by infiltration at 1E + 5 CFU/mL. A, Leaf appearance of Col-0 plants 3 d after bacterial infiltration. B, Bacterial population in Col-0 leaves at day 0 and day 3 after infiltration. C, Leaf appearance (abaxial sides) 3 d after infiltration with Pst DC3118. D, Bacterial populations at day 0 and day 3 after infiltration with Pst DC3118. Results displayed here (B and D) are means of four different leaves from four different plants, with ses indicated. Statistical differences are detected with two-tailed t test (***, P < 0.001).

Figure 5.

Role of NPR1 in plant response to COR-deficient or wild-type Pst DC3000 bacteria. A, Stomatal apertures in Col-0 and npr1-1 leaf peels incubated with Pst DC3118 or DC3000 at 1E + 8 CFU/mL. Results are displayed as means of 30 to 60 stomata with ses shown. B, Leaf appearance and bacterial populations 3 d after dip inoculation of Col-0 and npr1-1 plants with Pst DC3118 at 1E + 8 CFU/mL. C, Bacterial populations at day 3 in leaves of Col-0, npr1-1, and fls2 plants infiltrated with Pst DC3118 at 1E + 5 CFU/mL. B and C, Results are displayed as means of four different leaves from four different plants, with ses shown. Statistical differences are detected with two-tailed t test (***, P < 0.001) for A and B, and with ANOVA (P = 0.014) followed by Turkey's multiple comparison test (showing comparisons to Col-0; *, P < 0.05) for C.

Figure 6.

The involvement of GPA1 in response to COR-deficient or wild-type Pst DC3000 bacteria. Leaf appearance of (A) and bacterial populations in (B) Col-0 and gpa1-3 plants dip inoculated with Pst DC3118 at 1E + 8 CFU/mL. C, Stomatal apertures of Col-0 and gpa1-3 leaf peels treated with water and Pst DC3118 or DC3000 at 1E + 8 CFU/mL. D, Bacterial populations at day 0 and day 3 in leaves of Col-0 and gpa1-3 plants infiltrated with Pst DC3118 at 1E + 5 CFU/mL. Results are displayed as means with ses from four different leaves of four different plants for B and D, and of 30 to 60 stomata for C. Statistical differences are detected with two-tailed t test (***, P < 0.001).

Figure 7.

SA acts upstream of ABA in stomatal closure response. Data shown here are stomatal apertures of Col-0, eds5-1, sid2, and nahG leaf peels treated with buffer or ABA (10 μm; A); Col-0, eds5-1, sid2, and aba2-1 leaf peels treated with buffer or SA (20 μm; B); Col-0 and npr1-1 leaf peels treated with buffer or SA (20 μm; C); and Col-0, npr1-1, and fls2 leaf peels treated with buffer or ABA (10 μm; D). All results are displayed as means of 30 to 60 stomata, with ses indicated. Statistical differences between chemical treatments and buffer controls are detected with two-tailed t test (***, P < 0.001).

Figure 8.

Root elongation response to flg22 treatment. Data shown here are root lengths of wild-type plants Col-0 and Ler; mutant plants eds5-1 (SA synthesis), ost1-2 (ABA signaling, Ler background), aba2-1 (ABA synthesis), and gpa1-3 (α-subunit of the heterotrimeric G protein, ABA signaling) that were grown in liquid Murashige and Skoog medium for 10 d in the presence or absence of flg22 (10 μm). Results are displayed as means of 15 to 24 plants, with ses indicated. Statistical differences between treatments with or without flg22 are detected with two-tailed t test (***, P < 0.001).