Influence of <i>Verticillium longisporum</i> VL43 infection on protein content of whole leaf extracts, in apoplastic washing fluid and on the number of protein spots after 2-D electrophoresis.

<p>Plants were analyzed 25 days post infection. Protein spots were determined by Proteomweaver software in silver stained gels (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-g001" target="_blank">Fig. 1</a>). Data indicate means (N ± SE).</p

Fungal DNA, stunting of the rosette, and relative transcript abundance of genes encoding apoplastic proteins from <i>Arabidopsis thaliana</i> that were either induced or repressed by treatment with <i>Verticillium longisporum</i> (VL43).

<p>(A) Typical time course of stunting white symbols) and VL43 DNA (black symbols) in Arabidopsis rosettes (n≥4 biological replicates ± SE, except 10 dpi, where pooled samples were analyzed due limited material), T = traces of VL43 DNA detected. Stunting was determined as projected rosette area of VL infected plants/projected rosette area of mock inoculated plants. (B) Transcript levels of <i>CILLP</i>, <i>SCPL20</i>, <i>AGAL2</i> and <i>GLP3</i> and (C) transcript levels of the peroxidases <i>PRX52</i> (right axis), <i>PRX34</i>, <i>P37</i> and <i>PA2</i> (left axis). Transcript levels were normalized to actin and expressed as transcript abundance in VL-infected plants/transcript abundance in mock-inoculated plants (n≥6 biological replicates ± SE). *indicate significant differences between mock and VL-infected plants.</p

Proteins significantly affected in the apoplast of <i>Verticillilum longisporum</i> VL43-infected compared with that of mock-infected <i>Arabidopsis thaliana</i> leaves.

<p>*Factor: protein abundance in samples of VL-treated plants/protein abundance in mock-inoculated plants,</p><p>**92% homology to PRX At437520.</p><p>Only those spots were analyzed that were reproducibly observed in two independent experiments. In each experiment three biological replicates were analyzed. For further details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-t004" target="_blank">Table 4</a>. Spot identification numbers refer to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-g001" target="_blank">Figure 1</a>. Peptides are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone.0031435.s003" target="_blank">Table S1</a>.</p

Proteins identified by LC-MS/MS in the apoplast of <i>Arabidopsis thaliana</i> leaves.

<p>*encoded in the chloroplast genome.</p><p>Apoplastic washing fluids from mock-infected plants were separated by 2-D-gel electrophoresis, picked, trypsinated and used for determination fragment composition by LC-MS/MS. Identification was achieved by data bank queries as outlined under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#s4" target="_blank">materials and methods</a>. Spot numbers refer to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-g001" target="_blank">Fig. 1</a>. S, C, and M indicate predicted signal peptides for the secretory pathway, chloroplasts or mitochondria, respectively. RC indicates reliability classes for the prediction of localization (1 = high, 5 = low). Peptides are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone.0031435.s003" target="_blank">Table S1</a>.</p

Metabolic fingerprinting of AWFs of mock- and <i>Verticillium longisporum</i> VL43-infected <i>Arabidopsis thaliana</i> plants.

<p>AWF was obtained from <i>A. thaliana</i> leaves 21 days post infection and analyzed by UPLC-TOF-MS. (A) PCA plot of compounds in extracted AWFs measured in negative ionization mode. Samples from mock treated plants are framed in black whereas samples from infected plants are framed in red. (B) 1D-SOM matrix after metabolite-based clustering of the 1775 infection markers with <i>p</i><5×10⁻⁴. Data sets from positive and negative ionisation mode were combined. Red framed prototypes indicate markers with increased intensities after VL infection. The data set includes three biological replicates with three technical replicates per sample. The vertical axis represents the two experimental conditions. The horizontal axis corresponds to the cluster numbers. The intensities were normalized and colour coded according to the indicated scale.</p

Infection markers identified in leaves of the apoplast of <i>Arabidopsis thaliana</i> infected with <i>Verticillium longisporum</i> VL43.

<p>AWF was obtained from <i>A. thaliana</i> leaves 21 days post infection and analyzed by UPLC-TOF-MS. Data are means of 3 biological and 3 technical replicates. SI = Signal intensity, Mode of identification refers to A) Exact mass measurement, B) retention time (RT) comparison to authentic standards, C) UV/VIS spectrum, and D) MS/MS fragmentation. <i>P</i>-values were calculated by Students t-test.</p

<em>Verticillium longisporum</em> Infection Affects the Leaf Apoplastic Proteome, Metabolome, and Cell Wall Properties in <em>Arabidopsis thaliana</em>

<div><p><em>Verticillium longisporum</em> (VL) is one of the most devastating diseases in important oil crops from the family of Brassicaceae. The fungus resides for much time of its life cycle in the extracellular fluid of the vascular system, where it cannot be controlled by conventional fungicides. To obtain insights into the biology of VL-plant interaction in the apoplast, the secretome consisting of the extracellular proteome and metabolome as well as cell wall properties were studied in the model Brassicaceae, <em>Arabidopsis thaliana</em>. VL infection resulted in increased production of cell wall material with an altered composition of carbohydrate polymers and increased lignification. The abundance of several hundred soluble metabolites changed in the apoplast of VL-infected plants including signalling and defence compounds such as glycosides of salicylic acid, lignans and dihydroxybenzoic acid as well as oxylipins. The extracellular proteome of healthy leaves was enriched in antifungal proteins. VL caused specific increases in six apoplast proteins (three peroxidases PRX52, PRX34, P37, serine carboxypeptidase SCPL20, α-galactosidase AGAL2 and a germin-like protein GLP3), which have functions in defence and cell wall modification. The abundance of a lectin-like, chitin-inducible protein (CILLP) was reduced. Since the transcript levels of most of the induced proteins were not elevated until late infection time points (>20 dpi), whereas those of <em>CILLP</em> and <em>GLP3</em> were reduced at earlier time points, our results may suggest that VL enhances its virulence by rapid down-regulation and delay of induction of plant defence genes.</p> </div

<i>Verticillium longisporum</i> VL43 induced disease symptoms in <i>Arabidopsis thaliana</i>.

<p>Measurements were taken 24 or 25 days post inoculation on the indicated number (n) of plants. Data indicate means (± SE). Values of <i>p</i>≤0.05 indicate significant differences between mock-inoculated and <i>Verticillium longisporum</i> (VL)-infected plants.</p

Typical 2-D gels showing <i>Arabidopsis thaliana</i> apoplastic leaf proteins after mock (A) or <i>Verticillium longisporum</i>

<p><b>(VL43) infection (B).</b> Proteins were extracted 25 dpi and 80 µg were loaded for separation. Gels were stained with silver nitrate. Numbers in the gels indicate protein spots analyzed by ESI-LC/MS from preparative Coomassie-stained gels.</p

Fourier transform infrared spectra of cell walls of mock- and <i>Verticillium longisporum</i> VL43-infected <i>Arabidopsis thaliana</i> plants (A) and the difference spectrum (B).

<p>The difference spectrum was generated by subtracting the mean spectrum of cell walls from mock infected plants from that of VL-infected plants. Spectra were recorded with a resolution of 4 nm and are shown in the range from 800–1800 cm⁻¹; data were baseline corrected and vector normalized. Mean spectra were calculated (n = 8 per treatment) and subtracted.</p