Article Figures & Data
Figures
- Figure 1.
Long-chained oxo-C14-HSL has no impact on plant growth and is not transported within the plant. A to C, Col-0 plants were grown for 10 d on one-half MS medium and afterward transferred to one-half MS medium supplied with 6 μm oxo-C14-HSL, or 6 μm C6-HSL. A, Col-0 plants grown for 4 d on C6-HSL or oxo-C14-HSL, bar = 1 cm. B, Fresh weights (mg/plant) of plants treated with different AHLs, n ≥ 80. C, Root length of Arabidopsis plants on media supplied with different AHLs for 4 d, n ≥ 40, *P ≤ 0.05 (Student’s t test). D, Oxo-C14-HSL is not transported systemically throughout the plant. Six micromolar oxo-C14-HSL was added to the root medium for 3 d. Subsequently, fresh plant material was extracted in acetone and detection assays were performed by using the reporter bacterium E. coli Top10 pSB403 lxR+luxI::luxCDABE. [See online article for color version of this figure.]
- Figure 2.
C14-HSL derivatives induce resistance against P. syringae. A, Proliferation of Pst on 5-week-old Arabidopsis was analyzed at hpi as indicated. Plants were untreated (control) or pretreated with acetone or 6 μm oxo-C14-HSL, 3 d prior to infection with Pst bacteria. **P ≤ 0.005; ***P ≤ 0.0005. B, Colony forming units (cfu) of Pst harvested from 5-week-old Arabidopsis plants pretreated for 3 d with acetone, 6 μm oxo-C14-HSL, or 6 μm OH-C14-HSL, and subsequently inoculated with Pst bacteria for 96 h. a = P < 5.2E-6, b = P < 9.7E-11 (Student’s t test).
- Figure 3.
Oxo-C14-HSL induces resistance against G. orontii and B. graminis. Five-week-old Arabidopsis or 5-d-old barley plants were pretreated for 3 d with 6 μm oxo-C14-HSL in hydroponics cultures and then fungal conidia suspension was sprayed on their leaves. A to d, Proliferation of G. orontii on Arabidopsis leaves. A, Number of mycelia per leaf at 5 d post inoculation (dpi). Student’s t test performed on the raw data resulted in P = 0.33 (control versus acetone); P = 0.04 (control versus oxo); and P = 0.01 (acetone versus oxo). B to D, Mycelia formation on untreated Arabidopsis leaves (control; B), pretreated with acetone (C), or oxo-C14-HSL (D), shows no alternation in fungal development. E to H, Proliferation of B. graminis on barley. E, Percent of interaction sites with ESH, papillae, and HR, on first leaves at 5 dpi. F, ESH formation, haustorium (arrow). G, Papillae formation (arrowhead). H, HR. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005 (Student’s t test).
- Figure 4.
Oxo-C14-HSL does not induce resistance against B. cinerea or P. cucumerina BMM. Five-week-old Arabidopsis plants were untreated (A and E), pretreated with acetone (B and F), or 6 μm oxo-C14-HSL (C and G) for 3 d. Conidial suspensions were dropped onto detached leaves. Necrotic symptoms or lesion diameters were analyzed at 5 dpi. Classification of symptoms: I, healthy leaf; II, leaf necrotic at 25%; III, 50% of leaf surface show necrosis; IV, 75% necrotic surface; V, 100% of the leaf surface is necrotic. D, Necrotic symptoms caused by B. cinerea. H, Lesions caused by P. cucumerina BMM.
- Figure 5.
Response to flg22 in plants pretreated with AHL. A, Oxidative burst in response to 100 nm flg22. Production of H2O2 was measured using the luminol-based assay in detached leaf discs from soil-grown plants. Leaves were untreated (control), or pretreated with acetone or oxo-C14-HSL for 3 d before treatment with flg22. Arrow shows the time of flg22 addition. B, Time course of phosphorylation status of AtMPK6 and AtMPK3. Whole 2-week-old Arabidopsis Col-0 seedlings were pretreated with 6 μm oxo-C14-HSL for 3 d. Responses were analyzed in seedlings during minutes after treatment with 100 nm flg22. Top section shows an immunoblot with αpERK1/2 antibody, the bottom section shows Coomassie stain of the membrane. Sections below show immunoblots with the specific αMAPKs antibodies. C to F, Transcriptional regulation of WRKY and PR1 genes after pretreatment with oxo-C14-HSL. Total mRNA was extracted from 2-week-old Arabidopsis Col-0 seedlings, pretreated for 3 d with 6 μm AHL plants, and subsequent treatment with 100 nm flg22 at hour as indicated. qRT-PCR analysis was performed using WRKY- and PR1-specific primers (Supplemental Table S1).
- Figure 6.
AHL fails to modulate the response to flg22 in MAPK mutants. A, Time course of phosphorylation status of AtMPK3 and AtMPK6 in Col-0, mpk3, and mpk6. Whole 2-week-old mpk3, mpk6, or Col-0 seedlings were pretreated with 6 μm oxo-C14-HSL for 3 d. Responses were analyzed during minutes after treatment with 100 nm flg22. Top sections show an immunoblot with αpERK1/2 antibody, bottom sections show Coomassie stain of the membrane. B, Pst proliferation on mpk3 and mpk6 plants. Plants were pretreated as indicated for 3 d and spray inoculated with bacteria. Cfu numbers were analyzed after 48 h. Col-0 plants were used as a control. C to F, Transcriptional regulation of WRKY transcription factors after pretreatment with oxo-C14-HSL. Total mRNA was extracted from 2-week-old Arabidopsis mpk3 or mpk6 seedlings, pretreated for 3 d with 6 μm AHL plants, and subsequent treatment with 100 nm flg22 at hour as indicated. qRT-PCR analysis was performed using WRKY-specific primers (Supplemental Table S1). The fold induction was normalized to 0 h after flg22 treatment, note that mpk3 mutant has approximately 4 and 144 times lower basal level of WRKY22 and WRKY29 than Col-0 plants, respectively. The basal level of WRKY22 expression in mpk6 plants is slightly higher; the WRKY29 is 3 times lower than in Col-0 plants.
- Figure 7.
Oxo-C14-HSL promotes accumulation of ROS and induces HR rate after challenge with Pst in Arabidopsis plant. Arabidopsis plants were grown on soil and detached leaves were pretreated for 3 d with water (control; A, E, I, L), acetone (B, F, J, M), or 6 μm oxo-C14-HSL (C, G, K, N). Pretreated leaves were inoculated with Pst strain AvrRpt2 (avirulent) by spraying. Leaves were stained with DAB to visualize the accumulation of H2O2 (A–G), or with TB to visualize the dead cells (H–N). A to C, DAB stain of Arabidopsis leaves inoculated with Pst AvrRpt2 for 48 h. E to G show magnifications of respective A to C. Bar = 0.1 cm. D, Representation of DAB-positive stained clusters in Pst AvrRpt2-inoculated leaves. Pretreated leaves were infected with bacteria and stained with DAB at hpi as indicated. Number of stained clusters was calculated per cm2, 60 clusters were analyzed in three independent experiments. H, Numbers of TB-positive stained clusters in Pst AvrRpt2-inoculated leaves. Pretreated leaves were inoculated with bacteria and stained with TB at hpi as indicated. Clusters presence was calculated per cm2, 60 clusters were analyzed in three independent experiments. I to K, TB stain of Arabidopsis leaves inoculated with Pst AvrRpt2 for 48 h. L to N show respective magnifications of I to K. [See online article for color version of this figure.]
Additional Files
Supplemental Data
Supplemental Table and Figures
Files in this Data Supplement:
- Supplemental Data - Supplemental Figure 1
- Supplemental Data - Supplemental Figure 2
- Supplemental Data - Supplemental Figure 3
- Supplemental Data - Supplemental Figure 4
- Supplemental Data - Supplemental Figure 5
- Supplemental Data - Supplemental Figure 6
- Supplemental Data - Supplemental Figure 7
- Supplemental Data - Supplemental Figure 8
- Supplemental Data - Supplemental Table
