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Biology Articles » Mycology » EDR2 negatively regulates salicylic acid-based defenses and cell death during powdery mildew infections of Arabidopsis thaliana » Figures

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- EDR2 negatively regulates salicylic acid-based defenses and cell death during powdery mildew infections of Arabidopsis thaliana

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Figure 1 Macroscopic phenotypes of the edr2-6 mutant. (A, B) Uninfected plants at 25 d after germination; (A) wild type (B) edr2-6. (C, D, F-L) Three-week-old plants photographed at 7 dpi with G. cichoracearum (C) wild type, (D) edr2-6, (E) edr2-6 (1) The top-half or the (2) bottom-half of each leaf was covered with medical tape prior to inoculation. (F) NahG, (G) pad4-1, (H) edr2-6 NahG, (I) edr2-6 pad4-1, (J) edr2-6 coi1-1, (L) edr2-6 ein2-1.

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Figure 2 Microscopic visualization of fungal growth and cell death on leaves of 3-week-old plants at 7 dpi with G. cichoracearum. Leaves were stained with trypan blue. (A) wild type, (B) edr2-6, (C) edr2-6 coi1-1, (D) edr2-6 ein2-1, (E) edr2-6 NahG, (F) edr2-6 pad4-1. cp, conidiophores bearing asexual conidia; dc, dead cells; tr, trichome. Bar = 22 μm (A, B, D, F), 40 μm (C) and 26 μm (E).

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Figure 3 Hydrogen peroxide and callose accumulation in edr2-6. Three-week-old plants were inoculated with G. cichoracearum and stained for either hydrogen peroxide (A, B) or callose (C, D). (A, C) Col-0, (B, D) edr2-6. In D, callose outlines dead mesophyll cells in edr2-6. dc, dead cells; p, papilla. Bar = 22 μm.

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Figure 4 Response of edr2-6 to pathogens. (A) Leaves from plants 7 dpi with the barley pathogen, Blumeria graminis f.sp. hordei. (1) Col-0, inoculation density ~50 conidia per mm², (2) edr2-6, inoculation density ~1 conidia per mm², (3) edr2-6, inoculation density ~15 conidia per mm², (4) edr2-6, inoculation density ~50 conidia per mm². (B) Leaves at 2 dpi with P. syringae tomato DC3000. (1, 3, 5) Col-0, (2, 4, 6) edr2-6. Inoculation titers: (1,2) 10²cfu per ml; (3,4,) 10⁴cfu per ml; (5,6) 10⁸cfu per ml. (C) Leaves at 2 dpi with 10⁸cfu of P. syringae tomato DC3000 (avrRpt2). (1) Col-0, (2) edr2-6. Plants were 3-weeks old at the time of inoculation.

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Figure 5 The expression of PR1 is enhanced in edr2-6. (A) Northern blot showing PR1 expression at various times (in days) following inoculation of Col-0 or edr2-6 with G. cichoracearum. (B) PR1 expression in Col-0 and edr2-6 at 2 days following treatment with water (-), 0.3 mM BTH or 0.5 mM SA. (C) PR1 expression in Col-0 and edr2-6 at 48 hpi with 0, 10²or 10⁸cfu per ml of P. syringae pv tomato DC3000.

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Figure 6 Predicted EDR2 gene and protein structure. Intron-exon structure of the EDR2 gene. The regions of the gene corresponding to the PH, START and DUF1336 domains are indicated by lines and the site of the T-DNA insertion in the edr2-6 mutation is indicated by an arrow along with the ATG start codon and TAA stop codon.

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Figure 7 Binding of the EDR2 PH-domain to lipids. GST-tagged EDR2 PH domain was affinity purified and used to probe blots spotted with various lipids. The PH-GST fusion proteins were detected with an anti-GST antibody. PHD-1: wild-type PH-domain of EDR2; PHD-2: mutated PH-domain of EDR2 (F93S); GST: glutathione S-transferase negative control. Compounds spotted on the membrane: 1, lysophosphatidic acid; 2, lysophosphatidylcholine; 3, phosphatidyl inositol; 4, phosphatidylinositol 3-phosphate; 5, phosphatidylinositol 4-phosphate; 6, phosphatidylinositol-5-phosphate; 7, phosphatidyl ethanolamine; 8, phosphatidyl choline; 9, sphingosine-1-phosphate; 10, phosphadidylinositol-3,4-phosphate; 11, phosphadidylinositol-3,5-phosphate; 12, phosphatidylinositol-4,5-phosphate; 13, phosphadidylinositol-3,4,5-phosphate; 14, phosphatidic acid; 15, phosphatidyl serine; 16, blank

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Figure 8 EDR2 localizes to multiple subcellular compartments. (A) EDR2:HA:eGFP construct, CH2. Note, the eGFP sequence included the sequence for 10 Ala at the N-terminus (pink block). (B) EDR2:HA:GFP (labeled CH2) restores the edr2-6 mutant to disease susceptibility and suppresses the chlorosis and necrosis phenotype. For each genotype, two leaves from 3-week-old plants are shown at 7 dpi with G. cichoracearum. (C) EDR2:HA:eGFP was localized mainly to the endoplasmic reticulum as shown by the fluorescently-labeled reticulate net-like structure. EDR2:HA:eGFP also localized to the plasma membrane (arrows) and to endosomes (arrowheads). The upper two panels are from cotyledons of 7-day-old seedlings. Young, recently divided cells (asterisk) exhibited reduced EDR2:HA:eGFP fluorescence compared to more mature cells, including stomatal guard cells. The lower two panels are from leaves from 7-week old plants. St, stomata. Bars = 13.4 μm. (D) EDR2:HA:eGFP and the MitoTracker dye stain different sub-cellular structures. Merged image of the MitoTracker image (red) and the EDR2:HA:eGFP image (green). Thick arrows point to small bodies, mitochondria, stained with the MitoTracker dye and thin arrows point to endosomes tagged with EDR2:HA:eGFP. EDR2:HA:eGFP also localizes to the plasma membrane. Bars = 13.4 μm.