EDR1 negatively regulates the kinase activity of MPK3 and MPK6.

<p>(<b>A–B</b>) The transcript accumulation of <i>FRK1</i> was measured by quantitative real-time RT-PCR. Leaves were collected for RNA isolation at different time points after infection with <i>G. cichoracearum</i> (<b>A</b>) or <i>Pto</i> DC3000 (in 10 mM MgCl₂) (<b>B</b>). Error bars represent the standard deviation of three biological replicates. Asterisks indicate statistically significant differences (P<0.05, Student's <i>t</i>-test). (<b>C–D</b>) The plants were infected with <i>G. cichoracearum</i> (<b>C</b>) and <i>Pto</i> DC3000 (<b>D</b>), respectively. Immunoblotting was performed using an anti-phospho-p44/42 MAPK (Thr202/Tyr204) (anti-pTEpY) antibody. The large subunit of Rubisco is shown as a protein loading control. The experiment was repeated at least three times with similar results. PM: powdery mildew infection.</p

The <i>mpk3-1</i> mutation suppressed the <i>edr1</i> phenotype.

<p>(<b>A</b>) Immunoblot for Col-0, <i>edr1</i>, <i>mpk3-1</i> and <i>edr1 mpk3-1</i> was performed using specific anti-MPK3 antibody. The large subunit of Rubisco is shown as a protein loading control. (<b>B</b>) Col-0, <i>edr1</i>, <i>mpk3-1</i> and <i>edr1 mpk3-1</i> were grown in the greenhouse at 22°C and a 9 h light/15 h dark cycle. Pictures were taken after 5 weeks growth. (<b>C</b>) Plants were infected with <i>G. cichoracearum</i>. Pictures were taken at 7 dpi. (<b>D</b>) Powdery mildew infected leaves at 7 dpi were stained by trypan blue. Bar = 0.3 mm. (<b>E</b>) Quantification of fungal growth by counting the number of conidiophores per colony at 5 dpi. At least 30 colonies were counted for each sample. Error bars represent the standard deviation. Different letters represent statistically significant differences (P<0.05, one-way ANOVA). (<b>F</b>) Four-week-old plants of Col-0, <i>edr1</i>, <i>mpk3-1</i> and <i>edr1 mpk3-1</i> were treated with ethylene (100 µL/L) for three days in a sealed chamber. Pictures were taken after 3 days. (<b>G</b>) Chlorophyll content was measured in wild-type Col-0, <i>edr1</i>, <i>mpk3-1</i> and <i>edr1 mpk3-1</i> before and after treatment of ethylene (3 days). The ratio of chlorophyll content at day 3 to day 0 was calculated for each sample. Error bars represent the standard deviation of ten plants. Different letters represent statistically significant differences (P<0.05, one-way ANOVA). (<b>H</b>) Three-week-old Col-0, <i>edr1</i>, <i>mpk3-1</i> and <i>edr1 mpk3-1</i> plants were infected by <i>H. a.</i> Noco2. Spores were counted at 7 dpi. Error bars represent the standard deviation of three biological replicates. Different letters represent statistically significant differences (P<0.05, one-way ANOVA). The above experiments were repeated three times with similar results.</p

EDR1 interacts with MKK4 and MKK5.

<p>(<b>A</b>) <i>EDR1</i> full length (F), <i>EDR1</i> N-terminal domain (N) and <i>EDR1</i> C-terminal domain (C) were fused to the Gal4 DNA binding domain (BD). <i>MKK1</i>, <i>MKK2</i>, <i>MKK4</i>, <i>MKK5</i>, <i>MPK3</i> and <i>MPK6</i> were fused to the Gal4 transactivation domain (AD). Different pairs of constructs were cotransformed into yeast isolate AH109 to test the interaction. 10 µL suspension (OD₆₀₀ = 0.5) of each cotransformant was dropped on the synthetic dropout (SD) medium lacking Leu and Trp (left) and SD medium lacking Ade, His, Leu and Trp (right), respectively. Pictures were taken after 2 days incubation. (<b>B</b>) YFP^YN-fused <i>EDR1</i> and YFP^YC-fused <i>MKK4</i>/<i>MKK5</i> were co-expressed in <i>N</i>. <i>benthamiana</i>. YFP fluorescence was detected by confocal microscopy. Cotransformants of YFP^YN-EDR1 and YFP^YC, YFP^YN and YFP^YC-MKK4, or YFP^YN and YFP^YC-MKK5 were used as controls. Bar = 50 µm. (<b>C</b>) <i>EDR1</i> N-terminal domain was expressed alone or co-expressed with <i>MKK4</i> and <i>MKK5</i> in <i>N</i>. <i>benthamiana</i>. Proteins were extracted after 48 h, and subjected to immunoprecipitation by anti-HA antibody, followed by immunoblotting using anti-Myc and anti-HA antibodies, respectively. (<b>D</b>) <i>EDR1-Flag</i> transgenic plants and <i>EDR1-Flag</i>/<i>HA-MKK5</i> double transgenic plants were used for co-IP. The proteins were analyzed by immunoblotting using anti-Flag or anti-HA antibody, respectively. The above experiments were repeated three times with similar results.</p

EDR1 regulates the protein levels of MKK4 and MKK5.

<p>(<b>A</b>) GFP and Cherry fluorescence of seedlings of transgenic plants that express MKK4-GFP or MKK5-GFP alone or with EDR1-Cherry, was detected by confocal microscopy using the same parameters. Bar = 50 µm. (<b>B</b>) The GFP fluorescence intensity was quantified by using ImageJ software. 30 cells from 10 independent leaves of each transgenic plant were used for the quantification of the intensity of GFP fluorescence. The results are shown as a box plot graph. Asterisks represent statistically significant differences (P<0.05, Student's <i>t</i>-test). (<b>C</b>) Immunoblot was performed for each sample using anti-GFP antibody. The large subunit of Rubisco is shown as a protein loading control. (<b>D</b>) GFP fluorescence of seedlings of transgenic plants Col-0::<i>MKK5-GFP</i> and <i>edr1</i>::<i>MKK5-GFP</i> was detected by confocal microscopy using the same parameters. Bar = 50 µm. (<b>E</b>) Immunoblot was performed for Col-0::<i>MKK5-GFP</i> and <i>edr1</i>::<i>MKK5-GFP</i> using anti-GFP antibody. The large subunit of Rubisco is shown as a protein loading control.</p

<i>mkk4</i> and <i>mkk5</i> suppress <i>edr1</i>-mediated resistance to powdery mildew and cell death.

<p>(<b>A</b>) Col-0, <i>edr1</i>, <i>mkk4</i>, <i>edr1 mkk4</i>, <i>mkk5</i> and <i>edr1 mkk5</i> were grown in the greenhouse at 22°C and a 9 h light/15 h dark regime. Pictures were taken after 5 weeks of growth. (<b>B</b>) Plants were infected by <i>G. cichoracearum</i>. Pictures were taken at 7 dpi. (<b>C</b>) Powdery mildew infected leaves at 7 dpi were stained by trypan blue. Bar = 0.1 mm. (<b>D</b>) Fungal growth was assessed by counting the number of conidiophores at 5 dpi. At least 30 colonies were counted for each sample. Error bars represent the standard deviation. Different letters represent statistically significant differences (P<0.05, one-way ANOVA). (<b>E</b>) Plants were infected by <i>G. cichoracearum</i> for 3 days. Immunoblot was performed using anti-pTEpY antibody. The large subunit of Rubisco is shown as a protein loading control. The experiment was repeated three times with similar results.</p

EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

<div><p>Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that <i>edr1</i> mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the <i>mpk3</i>, <i>mkk4</i> and <i>mkk5</i> mutations suppress <i>edr1</i>-mediated resistance, and over-expression of <i>MKK4</i> or <i>MKK5</i> causes <i>edr1</i>-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.</p></div

The <i>exo70B1-3</i> mutants accumulate high levels of SA and their enhanced resistance to <i>G. cichoracearum</i> requires <i>EDS5, NPR1</i> and <i>PAD4</i>.

<p><b>(A)</b> Four-week-old plants were infected with <i>G. cichoracearum</i> at high inoculum densities, and representative leaves removed from plants at 7 dpi with <i>G. cichoracearum</i>. <b>(B)</b> Leaves removed from plants infected with <i>G. cichoracearum</i> at high inoculum densities at 7 dpi were stained with trypan blue to show hyphae and dead cells. Bar = 100 μm. <b>(C)</b> Fungal growth in plants infected with <i>G. cichoracearum</i> at low inoculum densities at 5 dpi was assessed by counting the number of conidiophores per colony. Lower-case letters indicate statistically significant differences (<i>p <</i> 0.05; one-way ANOVA). <b>(D)-(E)</b><i>exo70B1-3</i> mutants accumulate high levels of free SA. SA was extracted from leaves of uninfected four-week-old plants <b>(D)</b>, or leaves of four-week-old plants infected with <i>G. cichoracearum</i> at high inoculum densities at 3 dpi <b>(E)</b>. Bars represent mean and standard deviation from three biological replicates for each genotype. Lower-case letters indicate statistically significant differences (<i>p</i> < 0.05; one-way ANOVA). These experiments were repeated three times with similar results.</p

Defense responses of wild type, <i>exo70B1-3, pen1-1</i> and <i>pen1-1 exo70B1-3</i> plants to adapted and non-adapted powdery mildew pathogens.

<p><b>(A)-(C)</b> Four-week-old plants were infected with <i>G. cichoracearum</i>. The <i>pen1-1</i> mutation did not affect powdery mildew resistance in <i>exo70B1-3</i>. <b>(A)</b> Leaves at 7 dpi were removed and photographed. Bar = 10 mm. <b>(B)</b> Plant cell death and fungal growth were examined with trypan blue staining. Bar = 50 μm. <b>(C)</b> Fungal growth in plants was quantified by counting the number of conidiophores per colony at 5 dpi. Bars represent mean and sd (n = 30). Statistically significant difference from wild type is indicated by lower case letters (<i>p</i> < 0.05; one-way ANOVA). The experiments were repeated three times with similar results. <b>(D)</b> Four-week-old plants were infected with <i>Blumeria graminis f. sp. tritici</i>. Frequency of epidermal single cell death induced by infection with <i>Blumeria graminis f. sp. Tritici</i> was calculated at 1 and 2 dpi. The <i>pen1-1</i> mutant showed a significantly higher frequency of epidermal single cell death upon <i>Blumeria graminis f. sp. Tritici</i> infection; by contrast, the frequency of epidermal single cell death in <i>exo70B1-3</i> was similar to that of wild type. Bars represent mean and standard deviation (n = 6, scoring 40 sites per time point). Asterisks indicate statistically significant difference from wild type (<i>p</i> < 0.01; Student’s <i>t</i>-test). The experiments were repeated three times with similar results.</p

<i>exo70B1</i>-mediated resistance and cell death require the TIR-NBS protein TN2.

<p><b>(A-C)</b> Four-week-old plants were infected with <i>G. cichoracearum</i>.<b>(A)</b> Intact plant (upper panel) or representative leaves (lower panel) from the infected plants were photographed at 7 dpi. The <i>exo70B1-3</i> mutant was resistant, but the <i>exo70B1-3 tn2-1</i> mutant was susceptible to <i>G. cichoracearum</i>. Bar = 10 mm. <b>(B)</b> Leaves from infected plants were stained with trypan blue at 7 dpi. Many fungal spores were produced in wild type and <i>exo70B1-3 tn2-1</i>, but few spores were produced in <i>exo70B1-3</i>. Bar = 50 μm. <b>(C)</b> Fungal growth was assessed by counting the number of conidiophores per colony at 5 dpi. Bars represent mean and standard deviation from three independent biological replicates (n = 30). The asterisk indicates a statistically significant difference (<i>p</i> < 0.05; Student’s <i>t</i>-test). <b>(D)</b> Four-week-old plants of wild type, <i>exo70B1-3</i> and <i>exo70B1-3 tn2-1</i> were inoculated with <i>Pto</i> DC3000 at OD₆₀₀ = 0.0005. Bacterial growth was assessed at days 0 and 3. Bars represent means and standard deviation of three independent biological replicates. The asterisk indicates a statistically significant difference (<i>p</i> < 0.05, Student’s <i>t</i>-test). <b>(E)</b> Quantitative RT-PCR analysis of <i>PR1</i> expression in the infected plants with <i>G. cichoracearum</i>. The relative transcript levels were examined by real-time PCR and normalized to <i>ACTIN2</i>. Bars represent mean and standard deviation from three independent experiments. The asterisk indicates statistically significant difference (<i>p</i> < 0.05, Student’s <i>t</i>-test). <b>(F)</b> Protein structure of TN2. An arrowhead indicates the position of the mutated amino acid in each <i>tn2</i> mutant. <b>(G)-(H)</b> The transcript levels of <i>TN2</i> were examined by quantitative real-time PCR, and normalized to <i>ACTIN2</i>. Uninfected wild type and <i>exo70B1-3</i> plants (G) and wild type plants infected with <i>G. cichoracearum</i> (day 0: uninfected control) (H). Bars represent mean and standard deviation from three biological experiments. The asterisk indicates a statistically significant difference (<i>p</i> < 0.05, Student’s <i>t</i>-test).</p

EXO70B1 interacts with TN2.

<div><p><b>(A)</b> EXO70B1 interacted with TN2 in a yeast two-hybrid assay. Yeast cells containing the indicated plasmids were spotted onto the SD-His-Ura-Trp containing X-Gal. EV, empty vector; TN2-TIR, the TIR domain of TN2; TN2, full-length TN2; EXO70B1, full-length EXO70B1. Western blots verify the accumulation of Y2H fusion proteins. Expected sizes of pEG202:EV = 24 kD, pEG202:TN2-TIR = 48 kD, pEG202:TN2 = 67 kD, pJG4-5:EXO70B1 = 84 kD. Ponceau stain of membranes indicates equal loading.</p> <p><b>(B)</b> Interaction between EXO70B1 and TN2 was examined with a BiFC assay in <i>N. benthamiana</i>. EXO70B1 and TN2 were fused to the C-terminal or N-terminal fragment of YFP (cYFP or nYFP) respectively. Agrobacterium GV3101 strains carrying different cYFP and nYFP pairs were infiltrated into leaves of <i>N. benthamiana</i>. YFP fluorescence was observed only with the transiently expressed EXO70B1-cYFP and TN2-nYFP, but not in the negative controls in <i>N. benthamiana</i>. Bar = 50 μm.</p> <p><b>(C)</b> EXO70B1 interacted with TN2 in a Co-IP assay in <i>N. benthamiana</i>. <i>35Spro:EXO70B1-FLAG</i> was co-expressed with <i>35Spro:TN2-Myc</i> in <i>N. benthamiana</i> leaves. Total protein was extracted, and subjected to immunoprecipitation of TN2 protein by anti-Myc antibody. Proteins were analyzed by immunoblotting using anti-Myc or anti-FLAG antibody, as indicated.</p> <p>These experiments were repeated three times with similar results.</p></div