Disease symptoms in Arabidopsis WT plants and <i>at-mmp</i> mutants infected by the powdery mildew fungus <i>Golovinomycis orontii</i>.

<p>Four-week-old plants were spray-inoculated with 20–40×10³ conidia per ml suspension. Leaves were detached and photographed at 11 dpi. The amount of conidia per mg of leaf fresh weight was determined. At least 10 individually plants were treated in each line. The error bars indicate the standard error. Experiments were repeated three times with similar results. Significant differences are marked: *p < 0.05, ***p < 0.001 (Student’s t-Test).</p

At2-MMP, At3-MMP, and At5-MMP are involved in basal resistance to <i>Botrytis cinerea</i>.

<p>Detached leaves from 4-week-old Arabidopsis plants were inoculated with fungal conidia. Five μl spore suspension adjusted to 50,000 conidia per ml were placed on the middle vein. Disease symptoms were evaluated at 4 dpi. The image shows representative symptoms out of 12 leaves per line. Experiments were repeated three times with similar results. Shown is the mean ± SE of one experiment. Significant differences are marked: *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s <i>t</i>-Test).</p

Proteolytic activity of At2-MMP.

<p>(<b>A</b>) Intercellular washing fluid (IWF) was extracted from six-week-old Arabidopsis plants using an infiltration-centrifugation method. About 30 fully-expanded rosette leaves were detached from 10 plants, IWF from transgenic and control plants were produced, with their proteolytic activities determined by degradation of myelin basic protein (MBP) and fractionation on 20% SDS-PAGE. (<b>B</b>) Activity of the IWF from At2-MMPox plants was inhibited by EDTA. (<b>C</b>) Degradation of MBP by recombinantly produced Mat-MMP2 (predicted mature At2-MMP). The Mat-MMP2 protein starts from the amino acid D in the cysteine switch motif PRCGNPD and ends before the C-terminal transmembrane domain. Both Mat-MMP2 and proteins produced from empty vector (EV) transformed cells were harvested from the supernatant after three times sonication. MBP was incubated with or without the presence of Mat-MMP2 at 37^°C for 0 min and 30 min. Samples were separated by 20% SDS-PAGE.</p

Responses of <i>At-MMPs</i> upon <i>Botrytis cinerea</i> infection.

<p>(A) Expression profiles of <i>At-MMPs</i> in Arabidopsis accession Col-0 (WT) leaves in response to <i>Botrytis cinerea</i>. Five-week-old plants were inoculated with the fungus by placing 5 μl of spore suspension (2x10⁵ spores/ml in potato dextrose broth [PDB]) on the center of the rosette leaves. Mock treatment was performed with PDB. Total RNA was extracted from leaves at the indicated time points and used for RT-PCR. UBQ5 was used as an internal control. Experiments were independently repeated three times with similar results. (B) Molecular phylogenetic analysis of MMPs in Arabidopsis. The evolutionary history was inferred by Neighbor-Joining method using a conserved region spanning the cysteine switch and Met-turn motif of AtMMP protein sequences.</p

Expression profiles of <i>At2-MMP</i> and <i>At3-MMP</i> in hormone signaling mutants after <i>B</i>. <i>cinerea</i> infection.

<p>Five-week-old Arabidopsis WT plants, SA pathway mutants (<i>NahG</i>, <i>ics1</i>, <i>npr1-3</i>, JA pathway mutants (<i>jar1-1</i>, <i>jin1</i>, <i>npr1-1</i>) and ethylene pathway mutant (<i>ein2-1</i>) were inoculated with <i>B</i>. <i>cinerea</i> by placing 5 μl spore suspension (2x10⁵ spores/ml in PDB) or PDB (mock) on the center of the rosette leaves. Total RNA was extracted from leaves at the indicated time points and used for RT-PCR. UBQ5 was used as an internal control. Experiments were independently repeated three times with similar results. hat: hours after treatment, hai: hours after inoculation.</p

Structure and subcellular localization of At2-MMP.

<p>(<b>A</b>) Domain structure of an At2-MMP-GFP fusion construct. Numbers denote the position of amino acids (aa). SP: signal peptide; Pro: propeptide domain; CYS: cysteine switch; F: predicted furin-cleavage site; CAT: catalytic domain; ZBD: zinc-binding domain; G: putative GPI-anchor modification site; TM: transmembrane domain. The <i>Sal</i>I site (GTCGAC, aa 337–338) is downstream of the zinc-binding domain and upstream of the GPI-anchor modification site. The GFP coding sequence was integrated at the <i>Sal</i>I site. (<b>B</b>) Subcellular localization of At2-MMP upon transiently expression of 35S::At2-MMP2-GFP together with 35S::mCherry in Arabidopsis WT leaves. At2-MMP-GFP was detected 24 h after bombardment. Left panel: without plasmolysis; middle and right panel: five min after plasmolysis induced with 50% glycerol. Bar = 20 μm.</p

Additional file 5 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 5. Table S

Additional file 4 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 4. Table S

Additional file 3 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 3. Table S

Additional file 1 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 1. Figures S1 to Figures S