Silencing of <i>pox12</i> suppresses maize penetration resistance to the <i>Δpep1</i> mutant.

<p>(<b>A</b>) (left panel): Aniline blue staining of control plants (BMV-YFPsi) show formation of papillae at points of SG200Δpep1 penetration attempts. SG200Δpep1 is arrested directly upon penetration. (right panel): <i>pox12</i>-silenced (BMV-POX12si) maize plants infected with SG200Δpep1. Strain SG200Δpep1 successfully penetrates epidermal cells (arrows), shows cell to cell penetrations (arrow heads) and reaches the mesophyll layer (M, chevron) without eliciting visible plant defense responses. Bars: 10 µm (<b>B</b>) Quantification of intracellular hyphae length of <i>U. maydis</i> SG200Δpep1 on <i>pox12</i>-silencing plants compared to control plants. Silencing of <i>pox12</i> led to a significant, ∼10-fold increase in length of intracellular SG200Δpep1 hyphae. (<b>C</b>) <i>pox12</i> expression was quantified by quantitative real-time PCR using leaf samples of 8 independent <i>pox12</i>-silenced plants (BMV/POX12si) and 7 control plants (BMV/YFPsi) 48 h after <i>U. maydis</i> SG200Δpep1 infection (for details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002684#s4" target="_blank">methods</a> section). Relative expression of <i>pox12</i> in BMV/YFPsi control plants was averaged and set to 1. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p

Pep1 directly inhibits peroxidase activity.

<p>(<b>A</b>) Measurement of HRP activity using an <i>in vitro</i> DAB assay. Dark coloration indicates peroxidase activity, visualized by the precipitation of DAB. The addition of purified GFP as well as heat inactivated Pep1 (Pep1_IA) does not interfere with HRP activity. Addition of 5 µM native Pep1 results in reduced DAB precipitation, indicating suppressed HRP activity. (<b>B</b>) Quantification of DAB based HRP activity assay. Different concentrations of Pep1 were added to the assay solution at two different relevant pH values. Pep1 exhibits the ability of concentration dependent suppression of HRP activity at pH 6.5 and 7.5. (<b>C</b>) Far Western blot shows physical interaction of HRP with Pep1 (18.5 kDa) but not GFP (29.8 kDa) (upper panel). As a loading control, a separate gel was equally loaded and stained with coomassie blue (lower panel). (<b>D</b>) Peroxidase activity of maize apoplastic fluid was determined in a quantitative DAB assay. Recombinant native Pep1 and heat inactivated Pep1 (Pep1_IA) were added to the reaction in respective concentrations. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p

Inhibition of the elicitor triggered oxidative burst in maize leaves.

<p>(<b>A</b>) Luminol based readout to determine H₂O₂ production in maize leaf discs. The oxidative burst was elicited by the addition of chitosan (2.5 mg/ml) one minute after starting the measurement. Concentrations of recombinant Pep1, Pep1_IA and GFP proteins: 10 µM. (<b>B</b>) Quantification of elicitor triggered H₂O₂ production in maize leaf discs. The bars represent the integrated signal intensity of the average of 6 independent samples over the first 5 min after elicitation. (<b>C</b>) Quantification of chitosan induced H₂O₂ production in maize leaf discs based on xylenol orange staining. The peroxidase inhibitor SHAM (2 mM), NADPH-oxidase inhibitor DPI (5 µM) as well recombinant Pep1 protein (10 µM) cause a significant reduction of H₂O₂. Heat inactivated Pep1 (Pep1_IA) does not influence the elicitor triggered oxidative burst. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p

<i>In vivo</i> interaction of Pep1 with POX12.

<p>Confocal images in (A) and (B) show <i>N. benthamiana</i> epidermal cells expressing BiFC constructs. (<b>A</b>) A plant cell co-expressing pSPYCE_C and pSPYNE_Pep1. Blue and red channels show apoplastic co-localization of the respective signals. No complementation of fluorescence is observed in the YFP channel. (<b>B</b>) A cell co-expressing pSPYCE_POX12 and pSPYNE_Pep1. Both signals co-localize in the apoplast. The YFP channel exhibits YFP fluorescence with the same localization pattern indicating restoration of the YFP complex due to direct interaction of POX12 and Pep1. Bars: 25 µm. (<b>C</b>) Yeast-Two-Hybrid experiment confirming interaction of Pep1 and POX12. Mutation of the putative active site of POX12 (POX12_m) did not abolish interaction with Pep1.</p

Characterization of plant defense responses to Δ<i>pep1</i> infections.

<p>(<b>A</b>) Appressorium (arrow) and penetrating hypha (arrow heads) of SG200RFP 24 hpi. Aniline blue staining indicates callose deposition at point of penetration which is suppressed upon establishment of biotrophy and intracellular growth. (<b>B</b>) Penetration attempts of SG200Δpep1RFP 24 hpi. Staining reveals formation of papillae below appressoria (arrows) and surrounding short invading hyphae. White bars: 20 µm. (<b>C</b>) TEM analysis of SG200 hypha in maize epidermis cell, stained with cerium chloride (CeCl₃). The plant plasma membrane (arrow heads) surrounds the invading hypha (H). Magnification of the biotrophic interface shows no signs of CeCl₃ staining (inset). V: plant vacuole, C: plant cytosol, UCW: <i>Ustilago maydis</i> cell wall, PCW: plant cell wall, M: mitochondrium, chevrons: tonoplast. (<b>D</b>) TEM picture of SG200Δpep1 invasion hypha (H) in maize epidermis cell. CeCl₃ staining localizes at the biotrophic interface (arrows), revealing accumulation of ROS (magnification, upper right). Tonoplast (chevrons) is ruptured (arrow heads), indicating an induced hypersensitive response of the penetrated cell (inset, lower left). Black bars: 1 µm.</p

Scavenging of reactive oxygen species suppresses maize penetration resistance to the <i>Δpep1</i> mutant.

<p>(<b>A</b>) Aniline blue staining of SG200Δpep1RFP attempting to penetrate maize epidermis cell, 24 hpi. A papilla is formed at the point of penetration (arrow). (<b>B</b>) SG200Δpep1RFP penetrating the maize epidermis of plants treated with 5 µM ascorbate. Arrows mark penetration sites. The invading hyphae succeed in cell to cell penetrations (arrow heads); in some cases, proliferation of the hypha could be observed (chevron). (<b>C</b>) SG200Δpep1RFP on maize leaves expressing PIN1-YFP. Enhanced autofluorescence of penetrated cells as well as cells surrounding the penetration event indicate a HR reaction of the plant. (<b>D</b>) SG200Δpep1RFP is able to penetrate PIN1-YFP expressing maize leaves after the treatment with 5 µM ascorbate without eliciting enhanced autofluorescence. Bars: 20 µm. (<b>E</b>) Quantification of the length of intracellularly growing hyphae of SG200Δpep1 in maize leaves treated with 5 µM ascorbate compared to mock treated leaves. The addition of ascorbate leads to an average 6-fold increase of hyphal length. (<b>F</b>) Quantification of maize epidermal cells expressing visual signs of HR per penetration attempt by SG200Δpep1. The ascorbate treated plants show a ∼45% decrease in HR symptoms compared to the mock treated plants. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p

Regulation of JA and SA associated maize marker genes in response to <i>U. maydis</i> wild type versus SG200Δpep1 infections.

<p>Expression levels of SA/JA marker genes were determined by quantitative real-time PCR. The expression values represent three biological replicates and are shown relative to <i>GAPDH</i> expression in each sample. Leaf samples of mock, SG200 or SG200Δpep1 infected plants were taken after 2 dpi. Expression levels in mock infected plants were set to 1 and relative expression of marker genes was calculated for SG200 (light grey bars) and SG200Δpep1 (dark grey bars) samples. (<b>A</b>) Expression of SA marker genes <i>atfp4</i>, <i>pox12</i> and <i>pr1</i> 24 hours after infection with strain SG200Δpep1 or SG200, respectively. (<b>B</b>) Expression of JA marker genes <i>cc9</i> and <i>bbi</i> after infection with strain SG200Δpep1 or SG200, respectively. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p