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

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

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

<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

Data_Sheet_1.docx

<p>Systemic wound response (SWR), a well-characterized systemic signaling response, plays crucial roles in plant defense responses. Progress in understanding of the SWR in abiotic stress has also been aided by the researchers. However, the function of SWR in freezing stress remains elusive. In this study, we showed that local mild mechanical wounding enhanced freezing tolerance in newly occurred systemic leaves of wheat plants (Triticum aestivum L.). Wounding significantly increased the maximal photochemical efficiency of photosystem II, net photosynthetic rate, and the activities of the antioxidant enzymes under freezing stress. Wounding also alleviated freezing-induced chlorophyll decomposition, electrolyte leakage, water lose, and membrane peroxidation. In addition, wounding-induced freezing stress mitigation was closely associated with the ratio between reduced glutathione (GSH) and oxidized glutathione (GSSG), and the ratio between ascorbate (AsA) and dehydroascorbate (DHA), as well as the contents of total soluble sugars and free amino acids. Importantly, pharmacological study showed that wounding-induced freezing tolerance was substantially arrested by pretreatment of wheat leaves with the scavenger of hydrogen peroxide (H₂O₂) or the inhibitor of NADPH oxidase (RBOH). These results support the hypothesis that local mechanical wounding-induced SWR in newly occurred leaves is largely attributed to RBOH-dependent H₂O₂ production, which may subsequently induce freezing tolerance in wheat plants. This mechanism may have a potential application to reduce the yield losses of wheat under late spring freezing conditions.</p><p>Highlights:</p><p>In our previous research, we found that local mechanical wounding could induce freezing tolerance in the upper systemic leaves of wheat plants. Surprisingly, in this paper, we further demonstrated that local mechanical wounding could also increase freezing resistance in newly occurred leaves of wheat plants. RBOH mediated H₂O₂ and ascorbate–glutathione cycle participate in this systemic wound response.</p

ABA content is altered in STTM165/166 plants.

<p>(A) Quantitative RT-PCR analysis of the expression of genes involved in ABA conjugation and de-conjugation. (B) Quantitative RT-PCR analysis of <i>BG1</i> expression in various tissues of wild type and STTM165/166 plants. (C) Comparison of the ABA content between wild type and STTM165/166 plants.</p

The miR165/166 Mediated Regulatory Module Plays Critical Roles in ABA Homeostasis and Response in <i>Arabidopsis thaliana</i>

<div><p>The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of <i>ABI4</i>, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of <i>BG1</i>, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis.</p></div

STTM165/166 plants are hypersensitive to ABA during seed germination and early seedling development.

<p>(A) The phenotype of wild type and STTM165/166 plants grown on MS medium supplemented with 1.0 μM ABA. Seedlings were photographed 9 days after stratification. (B, C) Germination and cotyledon greening analysis of the STTM165/166 and wild type plants in response to different concentrations of ABA (0, 1.0, 1.5 and 2.0 μM). Germination was scored at 4 days and greening was scored at 9 days after stratification. Three independent experiments were performed, and >100 seeds for each treatment were used for each experiment. Values are means ± standard deviation. (D, E) Quantitative RT-PCR analysis of the expression of both mature miR165/166 and its targets at seedling stage. Three independent experiments were performed, and values are means ± standard deviation.</p