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 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 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

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

Effect of the <i>rsw7-lph</i> mutation on cellularization of the male gametophyte and endosperm.

<p>(<b>a</b>): Pollen produced by <i>rsw7-lph</i>/+ plants; scanning electron micrograph showing a mixture of normal (star) and shriveled or collapsed grains; scale bar equals 10 μm. (<b>b–e</b>): Cell division defects of <i>rsw-lph</i> male gametophytes. DAPI staining of normal pollen (<b>b</b>) reveals two small, brightly staining sperm cell nuclei and the large, less brightly staining nucleus of the vegetative cell; pollen of <i>rsw7-lph</i>/+ plants is frequently mono-nucleate (<b>c</b>), bi-nucleate (<b>d</b>), or collapsed with no detectable DNA (<b>e</b>); scale bar in (<b>e</b>) equals 10 μm. (<b>f, g</b>): Absence of a cellularized endosperm in <i>rsw-lph</i> seed. Confocal micrographs of Schiff-stained seed show the presence of cell walls in the endosperm of seed containing wild type embryos (<b>f</b>) but not in seed containing mutant embryos (<b>g</b>); scale bar in (<b>g</b>) equals 50 μm.</p

Morphological and ultra-structural characteristics of <i>tio</i> and <i>rsw</i> mutants.

<p>Left: Seedling morphology. Images of wild type (<b>a</b>) <i>tio-12</i> (<b>d</b>) and <i>rsw-lph</i> (<b>e</b>) seedlings; root hairs were contrasted with methylene blue; scale bar equals 1 mm. Center: Anatomy of embryos. Confocal micrographs of wild type (<b>b</b>), <i>tio-12</i> (<b>e</b>), and <i>rsw-lph</i> (<b>h</b>) embryos at the heart stage of development stained with Alexafluor 488 hydrazide; scale bar equals 30 μm. Right: Ultra-structure of embryonic cells. Transmission electron micrographs showing cells of wild type (<b>c</b>), <i>tio-10</i> (<b>f</b>) and <i>rsw-lph</i> (<b>i</b>) embryos; the arrowheads point to cell wall stubs, and the stars in (<b>c</b>) and (<b>f</b>) mark the nucleus; the side of panels equals 10 μm.</p

Range of cytokinesis-defective embryo phenotypes.

<p>Nomarski images of whole mount cleared immature seed; embryos at the heart stage of development are shown on the left and endosperm nuclei (arrowhead) on the right: (<b>a</b>) wild type; (<b>b</b>) <i>kn</i> mutant derived from line 4–43 (see text); enlarged cell caught in mitosis is boxed and shown magnified; (<b>c</b>) <i>kn keu</i> double mutant; three nuclei of normal size (stars) are visible; (<b>d</b>) phenotype produced by a weak <i>kiesel</i> allele; (<b>e</b>) double mutant phenotype produced by line 4–43; (<b>f</b>) mutant arrested at globular stage with no apparent cell division defects derived from line 4–43. Scale bar equals 50 μm.</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

Molecular cloning of <i>OPN/TIO</i> and <i>LPH/RSW7</i>.

<p>(<b>a</b>) Top: Genetic and physical map of the <i>OPN/TIO</i> region on the lower arm of chromosome 1. Numbers below the line indicate the number of recombination events between <i>opn/tio</i> mutations and the corresponding molecular markers (with italics referring to recombinants on the centromeric side, and the total number of meiotic events analyzed listed on the far right); two BAC clones (black bars) spanning the <i>OPN</i>/<i>TIO</i> transcription unit (arrow) are shown below the map. Bottom: Domain structure of the TIO protein. The N-terminus consists of a <i>FU</i>-type kinase domain (gray bar, with the ATP binding pocket in black); the C-terminus contains four repeat motifs, the first two of which share significant similarity with Armadillo and HEAT repeats (white boxes labeled “AH”), while the second two show borderline similarity to Armadillo repeats (“A”; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146492#pone.0146492.ref035" target="_blank">35</a>]); the <i>tio</i>-<i>12</i> allele (mutation <i>12–15</i>) harbors a glycine to aspartic acid substitution in the ATP-binding pocket (listed in the dashed box, invariant positions of the consensus sequence marked with a star); the PROSITE consensus (motif no. P00107) of this sequence signature is: [LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}-[LIVCAT]-{PD}-x-[GSTACLIVMFY]-x(5,18)-[LIVMFYWCSTAR]-[AIVP]-[LIVMFAGCKR]-K; the <i>tio-10</i> allele (mutation <i>OX10</i>) harbors a cystein to tyrosine substitution in a portion of OPN without significant similarity to known motifs. (<b>b</b>) Top: Genetic and physical map of the <i>LPH/RSW7</i> region on the lower arm of chromosome 2, organized as in (<b>a</b>). Bottom: Domain structure of the predicted RSW7 protein. The N-terminal catalytic core (grey bar) and the neck domain (black bar) show strong similarity to members of the kinesin-5 family; the insertion sites of the two T-DNA alleles, <i>rsw7-118</i> and <i>rsw7-92</i>, are marked with diamonds; the <i>rsw7</i>-<i>lph</i> allele harbors a glycine to arginine substitution in a conserved portion of the catalytic core; an alignment of this portion with human Eg5/KIF11 and <i>E</i>. <i>nidulans</i> BimC (GenBank accession nos. P52732, P17120) is shown in the dashed box, with stars representing invariant and colons conserved positions; folding of this domain is shown below the alignment and inferred from the crystal structure of rat brain kinesin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146492#pone.0146492.ref067" target="_blank">67</a>] (L12/MT2: loop 12, microtubule binding domain 2; α5: alpha helix 5, L13: loop 13, β8: beta sheet 8).</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