Volume 5, Issue 6, Pages 1389-1402 (November 2012) Transcriptional Activation and Production of Tryptophan-Derived Secondary Metabolites in Arabidopsis Roots Contributes to the Defense against the Fungal Vascular Pathogen Verticillium longisporum  Tim Iven, Stefanie König, Seema Singh, Susanna A. Braus-Stromeyer, Matthias Bischoff, Lutz F. Tietze, Gerhard H. Braus, Volker Lipka, Ivo Feussner, Wolfgang Dröge-Laser  Molecular Plant  Volume 5, Issue 6, Pages 1389-1402 (November 2012) DOI: 10.1093/mp/sss044 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Transcriptome Analysis of A. thaliana Roots 1 and 3 d Post V. longisporum Inoculation. (A, B) In vitro A. thaliana cultivation system for root inoculation with Vl43 conidia suspension. The roots of 14-day-old A. thaliana seedlings were inoculated by applying 500 μl 4 × 105 conidia ml−1 V. longisporum conidia suspension to the infection-canal that is cut to the solid growth media prior to sowing the Arabidopsis seeds. (C–E) Microscopic analysis of a spore germination time course of GFP-tagged Vl43 inoculated on A. thaliana roots 1 (C), 2 (D), and 3 (E) dpi. (F) Experimental set-up of the microarray experiment comparing the time points 1 and 3 dpi with respective mock-inoculated roots. The arrows represent the six Cy3/Cy5-labeled microarray slides. Arrow base and arrowheads represent the CY5 and Cy3-labeled samples, respectively. (G) Venn-diagram visualizing the overlap of differentially expressed genes in infected roots 1 and 3 dpi with V. longisporum. The diagram is showing the genes that are differentially expressed in infected roots (more than twofold, log2) compared to mock-inoculated roots (P < 0.1). (H) Results of FatiGO gene ontology (GO) enrichment analysis (Al-Shahrour et al., 2004) of the up-regulated genes 3 dpi (List1) compared to the set of array spotted genes of the Genome Oligo Set version 3.0 (List2) depicting the 16 significantly (P < 0.05) enriched terms for the biological process GO category of level 6 to level 9. The first row (Term) lists the significantly overrepresented GO terms. The number of annotated genes of the respective GO-term of each list is shown in the rows List1 positives and List2 positives. The table is sorted by decreasing odds ratio log values, which is a measure of enrichment of List1 compared to List2. Higher positive values represent higher enrichment of List1 in the respective GO term. FatiGO used 342 genes of List1 and 25885 genes of List2 after duplicate removal. 154 genes of list1 (45%) and 9233 genes of list2 (36%) could be annotated to GO terms of levels 6 to 9. (I) The amounts of the hormones indicated were measured in uninfected (gray) or infected (black) Arabidopsis roots 2, 4, 6, and 8 dpi. Given are hormone amounts calculated as nmol per g fresh weight (f.w.). Molecular Plant 2012 5, 1389-1402DOI: (10.1093/mp/sss044) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Coordinated Transcriptional Induction of the Tryptophan-Derived Secondary Metabolism. (A) Schematic representation of the biosynthetic pathway leading to tryptophan (light green), IGS (blue), and camalexin (dark green). Depicted are the major intermediates and enzymes. The V. longisporum-induced genes and the corresponding Arabidopsis Gene Identifier (AGI) are given in brackets. The (log2) induction values from the microarray experiment are shown in red square brackets for the 3-dpi time point. Metabolites found to be increased after infection are marked by a blue arrow. IAN, Indole-3-acetonitrile; I3CHO, indole-3-carbaldehyde; I3CA, indole-3-carboxylic acid; DHCA, dihydrocamalexic acid; I3G, indole-3-ylmethylglucosinolate; I3A, indole-3-ylmethylamine; 4MI3G, 4-methoxyindole-3-ylmethylglucosinolate; RA, raphanusamic acid. (B) qPCR analysis of relative transcript abundance in V. longisporum-infected A. thaliana Col-0 Wt roots. Expression of PAT1 (At5g17990), TSA (At4g02610), and ASA1 (At5g05730) was determined 2, 4, 6, and 8 dpi. The relative transcript abundance was normalized to mock-inoculated control (set to one). The mean values (+SE) are obtained from two biological and four technical replicates. For each replicate and condition RNA from pooled roots of three Petri dishes was prepared (>100 seedling roots). Molecular Plant 2012 5, 1389-1402DOI: (10.1093/mp/sss044) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 V. longisporum-Induced Expression of CYP79B2 and CYP79B3. (A) qPCR analysis of relative transcript abundance in V. longisporum-infected A. thaliana Col-0 Wt roots. Expression of CYP79B2 (At4g39950) and CYP79B3 (At2g22330) was determined 2, 4, 6, and 8 dpi. The relative transcript abundance was normalized to mock-inoculated control (set to one). The mean values (+SE) are obtained from two biological and four technical replicates. For each replicate and condition, RNA from pooled roots of three Petri dishes was prepared (>100 seedling roots). (B) Local expression of ProCYP79B2:LUC was monitored in mock-inoculated roots (left) and V. longisporum-inoculated roots (right) 3 dpi in stable A. thaliana reporter plants. The upper part shows the in vitro cultivated plants whereas the lower pictures provide overlays of the black/white and the luminescence exposures. Luminescence intensities are depicted in false color with an intensity gradient from blue to red representing low to high luminescence intensities, respectively. (C) Symptom development in Col-0 Wt (upper part) and cyp79b2 cyp79b3 mutant plants (lower part) of a representative infection experiment with 16 replicate plants per treatment 20 dpi. V. longisporum-inoculated plants (left) are compared with mock-inoculated control plants (right). (D) Quantification of V. longisporum-induced reduction of leaf area 20 dpi by determination of relative leaf area in Col-0 (Wt) and cyp79b2 cyp79b3 mutant plants. The relative leaf area represents the ratio of the leaf areas of V. longisporum-inoculated to mock-inoculated control plants where the leaf area of the control treatment was set to 100%. The mean values (+SD) are obtained from three biological replicates of typical experiments including 16 plants for each treatment. (E) PCR quantification of Verticillium sp. fungal DNA content in rosette plant tissue of root dip-inoculated A. thaliana plants normalized to 108 molecules of plant actin molecules. Shown is the mean V. longisporum DNA content of Col-0 Wt and cyp79b2 cyp79b3 mutant line 20 dpi. The mean values (+SD) are obtained from three biological replicates. The asterisk represents significant difference in V. longisporum DNA content between cyp79b2 cyp79b3 mutant and Col-0 Wt (* Student’s t-test, P < 0.05). Molecular Plant 2012 5, 1389-1402DOI: (10.1093/mp/sss044) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Induction of Indole Glucosinolate (IGS) Metabolism in A. thaliana Roots upon V. longisporum Inoculation. (A) qPCR analysis of relative transcript abundance in V. longisporum-infected A. thaliana Col-0 Wt roots. Expression of PEN2 (At2g44490), SUR1 (At2g20610), SUR2 (At4g31500), CYP81F2 (At5g57220), and PEL1 (At3g60120) was determined at the time points indicated. The relative transcript abundance was normalized to mock-inoculated control (set to one). The mean values (+SE) are obtained from two biological and four technical replicates. For each replicate and condition, RNA from pooled roots of three Petri dishes was prepared (>100 seedling roots). (B) HPLC–MS2 Quantification of RA in uninfected (gray) or Vl43-infected (black) Arabidopsis roots using the in vitro infection system. Given are amounts in nmol per g fresh weight (f.w.) measured 2, 4, 6, and 8 dpi. The mean values (+SD) are obtained from three biological replicates. For each replicate and condition, extracts from pooled roots of three Petri dishes were measured (>100 seedling roots). (C) Reduction of leaf area 20 dpi. The relative leaf area of Col-0 (Wt), pad3, cyp81f2, pen2-like (pel1), and cyp79b2 cyp79b3 mutant plants was determined. The relative leaf area represents the ratio of projected leaf areas of Vl43-inoculated compared to mock-inoculated control plants where the leaf area of the control treatment was set to 100%. The mean values (+SD) are obtained from three biological replicates of typical experiments including 16 plants for each treatment. (D) PCR quantification of Verticillium sp. fungal DNA content in rosette plant tissue of root dip-inoculated A. thaliana plants normalized to 108 molecules of plant actin molecules. Shown is the mean V. longisporum DNA content of Col-0 (WT), pad3, cyp81f2, pen2-like, and cyp79b2 cyp79b3 mutant plants 20 dpi. The mean values (+SD) are obtained from three biological replicates. The asterisk represents significant difference in Verticillium DNA content between the cyp79b2 cyp79b3 mutant and WT (* Student’s t-test, P < 0.05). The bars are shaded as depicted in the figure inlet. Molecular Plant 2012 5, 1389-1402DOI: (10.1093/mp/sss044) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Analysis of Camalexin and Indole-3-Carboxylic Acid (I3CA) Production and Effects in Arabidopsis–V. longisporum Interactions. (A) qPCR analysis of relative transcript abundance in V. longisporum-infected A. thaliana Col-0 Wt roots. Expression of CYP71A13 (At2g30770), CYP71A12 (At2g30750), and CYP71B15/PAD3 (At3g26830) was determined at the time points indicated. The relative transcript abundance was normalized to mock-inoculated control (set to one). The mean values (+SE) are obtained from two biological and four technical replicates. For each replicate and condition, RNA from pooled roots of three Petri dishes was prepared (>100 seedling roots). The bars are shaded as depicted in the inlet figures. (B) HPLC quantification of camalexin and HPLC–MS2 quantification of I3CA accumulation in mock treated (gray) and Vl43-infected (black) Wt root tissue given in nmol × g fresh weight−1. The mean values (+SD) were obtained from two and three biological replicates, respectively. (C) Growth inhibition of V. longisporum by camalexin or I3CA. Droplets of V. longisporum conidiospores of 0.4-cm diameter were placed on potato dextrose agar containing 0, 10, 100, 150, and 200 μM of camalexin or I3CA. The colony diameter was assayed after 2, 4, and 6 d. Molecular Plant 2012 5, 1389-1402DOI: (10.1093/mp/sss044) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions