Article Figures & Data
Figures
- Figure 1.
Atdpl1 mutants are more tolerant to B. cinerea but more susceptible to Pst than the wild type. B. cinerea conidia suspension was deposited by using drop inoculation (A and B) or spray inoculation (E) on leaves of wild-type (WT) and Atdpl1 mutant plants. Pst solution was infiltrated into wild-type and Atdpl1 mutant leaves (A, C, and D). A, Photographs represent disease symptoms observed 60 or 72 h after infection by the fungus or Pst, respectively. B, Symptoms due to B. cinerea infection were scored by defining three lesion diameter (d; in mm) classes. Statistical differences of the mean lesion diameters between wild-type and Atdpl1 plants were calculated with a Kruskal-Wallis test: **, P < 0.01; and ***, P < 0.005. C and D, Bacterial growth of virulent Pst strain DC3000 (C) and avirulent Pst strain AvrRPM1 (D) at 0, 6, 24, 48, and 54 hpi. E, B. cinerea and Pst growth was quantified by qRT-PCR 3 and 48 h after pathogen infection in leaves of wild-type and Atdpl1 mutant plants. Asterisks indicate significant differences between wild-type and Atdpl1 samples according to Student’s t test: ***, P < 0.005. Results are representative of three independent experiments.
- Figure 2.
Free LCB and LCB-P accumulation after challenge with pathogen. Leaves of wild-type (WT) or Atdpl1-1 mutant plants were sprayed with B. cinerea spore suspension (Bc) or potato dextrose broth (PDB; Control; A, B, E, and F) or infiltrated with Pst DC3000, Pst AvrRPM1, or MgCl2 (Control; C, D, G, and H). Quantifications of LCBs (A–D) and LCB-Ps (E–H) were performed 48 hpi. Asterisks on wild-type bars indicate significant differences between the pathogen-treated wild-type sample and the control sample, and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated wild-type sample and the pathogen-treated Atdpl1-1 sample according to Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.005. Results are means of four to five independent biological experiments ± sd. Notice the different scale of LCB-P levels between wild-type and Atdpl1-1 plants. DW, Dry weight.
- Figure 3.
GIPC contents after B. cinerea or Pst infection. Leaves of wild-type (WT; left) or Atdpl1-1 mutant (right) plants were sprayed with PDB (Control; A and B) or B. cinerea spore suspension (Bc; C and D) or infiltrated with MgCl2 (Control; E and F), Pst DC3000 (G and H), or Pst AvrRPM1 (I and J). Quantifications were performed 48 hpi. Asterisks on wild-type bars indicate significant differences between the pathogen-treated wild-type sample and the control sample, and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated wild-type sample and the pathogen-treated Atdpl1-1 sample according to Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.005. Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four to five independent biological experiments ± sd. DW, Dry weight.
- Figure 4.
Cer species produced by wild-type and Atdpl1-1 mutant plants upon pathogen infection. Leaves of wild-type (WT; left) or Atdpl1-1 mutant (right) plants were sprayed with PDB (Control; A and B) or B. cinerea spore suspension (Bc; C and D) or infiltrated with MgCl2 (Control; E and F), Pst DC3000 (G and H), or Pst AvrRPM1 (I and J). Quantifications were performed 48 hpi. Asterisks on wild-type bars indicate significant differences between the pathogen-treated wild-type sample and the control sample, and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated wild-type sample and the pathogen-treated Atdpl1-1 sample according to Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.005. Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four to five independent biological experiments ± sd. DW, Dry weight.
- Figure 5.
hCer species produced by wild-type and Atdpl1-1 mutant plants upon pathogen infection. Leaves of wild-type (WT; left) or Atdpl1-1 mutant (right) plants were sprayed with PDB (Control; A and B) or B. cinerea spore suspension (Bc; C and D) or infiltrated with MgCl2 (Control; E and F), Pst DC3000 (G and H), or Pst AvrRPM1 (I and J). Quantifications were performed 48 hpi. Asterisks on wild-type bars indicate significant differences between the pathogen-treated sample and the control sample, and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated wild-type sample and the pathogen-treated Atdpl1-1 sample according to Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.005. Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four to five independent biological experiments ± sd. DW, Dry weight.
- Figure 6.
Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation. Conductivity (μS cm−1) is shown for solution containing leaf discs from either the wild type (WT) or the Atdpl1-1 mutant inoculated with B. cinerea (Bc) or PDB (Control) solution (A) or Pst DC3000, Pst AvrRPM1, or 10 mm MgCl2 (B). Each value represents the mean ± sd of three replicates per experiment. The experiment was repeated three times with similar results.
- Figure 7.
Time course of PCD marker gene expression after B. cinerea or Pst infection. Leaves were sprayed with B. cinerea spore suspension (Bc) or PDB (Control; A, C, and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control; B, D, and F). The mean values ± sd from one representative experiment are shown. qRT-PCR of FMO (A and B), SAG13 (C and D), and SAG12 (E and F) expression was performed in wild type (WT) and Atdpl1-1 mutant plants with five biological replicates with comparable results.
- Figure 8.
Transient ROS production in response to pathogen infection in wild-type and Atdpl1-1 mutant plants. The time course of ROS production in wild type (WT) and Atdpl1-1 mutant plants is shown in response to B. cinerea (Bc; A), Pst DC3000 (B), or Pst AvrRPM1 (C) infection. Leaf discs were immersed in a solution containing either 105 spores mL−1 B. cinerea or 108 cfu mL−1 Pst. Error bars represent se from 12 biological repetitions. Three independent experiments were performed with similar results. RLUs, Relative light units.
- Figure 9.
Exogenous effects of t18:0-P and d18:0 on electrolyte leakage in response to pathogen infection in wild-type plants. A and B, B. cinerea conidia suspension was deposited on leaves of wild-type and Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d18:0 solution. Pst and either t18-0-P or d18:0 solution were coinfiltrated into wild-type and Atdpl1-1 leaves. Photographs represent symptoms observed 60 or 72 h after infection by the fungus or Pst, respectively. C to F, Conductivity (μS cm−1) of solution containing t18:0-P- or d18:0-infiltrated leaf discs from the wild type inoculated by spraying B. cinerea (Bc) or PDB (Control) solution (C and D) or by infiltration of Pst DC3000, Pst AvrRPM1, or 10 mm MgCl2 (E and F). Each value represents the mean ± sd of three replicates per experiment. The experiment was repeated three times with similar results.
- Figure 10.
Exogenous effects of t18:0-P and d18:0 on ROS production in response to pathogen infection in wild-type plants. The time course of ROS production in t18:0-1-P- or d18:0-treated wild-type plants is shown in response to B. cinerea (Bc; A and D), Pst DC3000 (B and E), or Pst AvrRPM1 (C and F) infection. Leaf discs were immersed in a solution containing 100 µm t18:0-1-P or d18:0 and either 105 spores mL−1 B. cinerea or 108 cfu mL−1 Pst. Error bars represent se from 12 biological repetitions. Three independent experiments were performed with similar results. RLUs, Relative light units.
- Figure 11.
Expression levels of JA and SA pathway-associated genes in wild-type (WT) and Atdpl1-1 mutant plants during B. cinerea (Bc) infection. Results are expressed as the fold increase in transcript level compared with the untreated control (0 h), referred to as the 1× expression level. Values shown are means ± sd of duplicate data from one representative experiment among five independent repetitions.
- Figure 12.
Expression levels of JA and SA pathway-associated genes in wild-type (WT) and Atdpl1-1 mutant plants during Pst infection. Results are expressed as the fold increase in transcript level compared with the untreated control (0 h), referred to as the 1× expression level. Values shown are means ± sd of duplicate data from one representative experiment among five independent repetitions.
- Figure 13.
Analysis of phytohormone accumulation in stressed wild-type and Atdpl1-1 mutant plants. JA, JA-Ile, and SA accumulation is shown in wild-type (WT) and Atdpl1-1 mutant plants 0 or 30 h following B. cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection. Asterisks indicate significant differences between wild-type and Atdpl1-1 samples according to Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.005. Values shown are means ± sd from one representative experiment among five independent repetitions. FW, Fresh weight.
- Figure 14.
Schematic overview of interconnections between sphingolipid metabolism, cell death, and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack. Upon disruption of the AtDPL1 gene, infected plants accumulate some LCB-P, hCer, and GIPC species, thus reducing cell death. In the Atdpl1 mutant, sphingolipid metabolism may also indirectly modulate cell death through its tight connection (double-headed dashed arrow) as a positive and/or negative regulator to jasmonate and/or SA signaling pathways, respectively. Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B. cinerea but more susceptible to Pst. T-bars indicate inhibition; single-headed arrows indicate activation; double-headed arrows indicate unknown regulatory mechanisms. Ald, Aldehyde; Ethan-P, phosphoethanolamine; LCBK, LCB kinase; LCB-P Pase, LCB-P phosphatase; LOH, LAG ONE HOMOLOG; SPHK1, SPHINGOSINE KINASE1.
Additional Files
Supplemental Data
Supplemental Figures and Table
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-4
- Supplemental Data - Supplemental Table
