Volume 9, Issue 5, Pages 682-695 (May 2016) Regulation of Pathogen-Triggered Tryptophan Metabolism in Arabidopsis thaliana by MYB Transcription Factors and Indole Glucosinolate Conversion Products  Henning Frerigmann, Mariola Piślewska-Bednarek, Andrea Sánchez-Vallet, Antonio Molina, Erich Glawischnig, Tamara Gigolashvili, Paweł Bednarek  Molecular Plant  Volume 9, Issue 5, Pages 682-695 (May 2016) DOI: 10.1016/j.molp.2016.01.006 Copyright © 2016 The Author Terms and Conditions

Figure 1 Indole Glucosinolate, Camalexin, and Indole-Carboxylic Acid Biosynthesis Are Closely Connected to Each Other. Solid lines indicate single enzymatic steps, whereas dashed arrows stand for several enzymatic steps. Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions

Figure 2 Expression of MYB51 and MYB122 Genes Is Induced by MAMP Application or Pathogen Challenge. Expression levels of genes encoding indole glucosinolate-related MYB transcription factors in Col-0 seedlings 12 h after flg22 treatment (A) and leaves of Col-0 plants 24 h after inoculation with spores of the necrotrophic pathogen Plectosphaerella cucumerina (B). Results are means ± SE from three/two independent experiments, each with three biological replicates (n = 9/6). Values marked with asterisks are significantly different from Col-0 (Student's t-test; *p < 0.05). Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions

Figure 3 flg22-Triggered Induction of Genes Linked with Trp Metabolism Is Partially Dependent on MYB Transcription Factors and PEN2 Myrosinase. Liquid-grown Arabidopsis seedlings were challenged with flg22 to identify the impact of pen2 and different myb mutations on the expression of genes required for IAOx (CYP79B2 [A], CYP79B3 [B]), IG (CYP83B1 [C]) as well as ICA and camalexin (CYP71A13 [D], CYP71B6 [E], and CYP71B15/PAD3 [F]) biosynthesis. Gene expression levels were assayed 12 h post inoculation. Results are means ± SE from three independent biological replicates (n = 10). Values marked with asterisks are significantly different from Col-0 (Dunnett's test for multiple comparisons; *p < 0.05). Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions

Figure 4 MYB34, MYB51, MYB122, and PEN2 Contribute to flg22-Triggered Accumulation of Trp-Derived Metabolites in Arabidopsis Seedlings. Accumulation of selected Trp-derived metabolites and raphanusamic acid, a PEN2 pathway side product, measured in Col-0 and indicated mutant seedlings 24 h after flg22 challenge. FW, fresh weight. Presented results are means ± SD from two independent experiments with four biological replicates in each (n = 8). Values marked with asterisks are significantly different from Col-0 (Dunnett's test for multiple comparisons; *p < 0.05). Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions

Figure 5 trans-Activation Assay of Camalexin and Glucosinolate Biosynthesis Promoters with MYB Transcription Factors. Transcription factor co-expression assay to determine the DNA binding and trans-activation potential of MYB34, MYB51, and MYB122 toward target promoters of the camalexin and ICA biosynthesis pathway genes CYP71A13 and CYP71B15/PAD3 in comparison with the indolic glucosinolate pathway gene CYP83B1. The promoters of CYP71A13, CYP71B15/PAD3, and CYP83B1 were fused to the uidA (GUS) reporter gene. Cultured A. thaliana cells were inoculated with the supervirulent Agrobacterium tumefaciens containing either only the reporter construct or the reporter construct in addition to Pro35S:MYB34, Pro35S:MYB51, or Pro35S:MYB122. The GUS activity indicates trans-activation of a promoter by an effector. The figure shows the relative GUS activity for the substrate methylumbelliferone in equal amounts of protein extract from these cells. Results are means ± SE from three independent biological replicates (n = 3). Values marked with asterisks are significantly different from Col-0 (Student's t-test; *p < 0.05). Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions

Figure 6 Expression of Genes Linked with the Biosynthesis of Indolic Phytoalexins upon P. cucumerina Infection Is Independent of the MYB Transcription Factors. The leaves of 5-week-old plants were infected with P. cucumerina to determine the impact of pen2 and different myb mutants on the expression of IAOx (CYP79B2 [A], CYP79B3 [B]), IG (CYP83B1 [C]), as well as ICA and camalexin (CYP71A13 [D], CYP71B6 [E], and CYP71B15/PAD3 [F]) biosynthesis. Gene expression levels were assayed 24 h post inoculation. Results represent means ± SE (n = 3) Values marked with asterisks are significantly different from Col-0 (Dunnett's test for multiple comparisons; *p < 0.05). Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions

Figure 7 MYB34, MYB51, and MYB122 Are Dispensable for Pathogen-Triggered Biosynthesis of Indole-3-Carboxylic Acids and Camalexin. Accumulation of selected Trp-derived metabolites measured in leaves of Col-0 and indicated mutant plants inoculated with the adapted strain of the fungal pathogen P. cucumerina. FW, fresh weight. Presented results are means ± SD from two independent experiments with three biological replicates in each (n = 6). Values marked with asterisks are significantly different from Col-0 (Dunnett's test for multiple comparisons; *p < 0.05). Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions

Figure 8 myb34/51/122 Plants Are pen2-like Susceptible to P. cucumerina. Plants of the tested genotypes were spray inoculated with spores of the indicated P. cucumerina isolates. (A) Average disease rating (DR ± SD, n = 15) of the indicated genotypes. DR varies between 0 (no symptoms) and 5 (dead plant). dpi, days post infection. Values marked with asterisks are significantly different from Col-0 (Dunnett's test for multiple comparisons; *p < 0.01). (B) Disease symptoms observed 5 dpi. (C) Real-time PCR quantification of fungal DNA (Pc β-tubulin) at 5 dpi. Values (SDs) are represented as the average of the n-fold fungal DNA levels, relative to the WT (Col-0) plants. The letters indicate significantly different statistical groups of genotypes (ANOVA; p < 0.05, Bonferroni's test). (D) Trypan blue staining of leaves collected at 2 dpi. Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions

Figure 9 Updated Model of Trp-Metabolism Regulation upon flg22 Treatment and P. cucumerina Inoculation. The three MYB TFs directly regulate transcription of genes linked with I3M biosynthesis, but not genes encoding enzymes involved in I3M modification or indolic phytoalexin biosynthesis (Frerigmann et al., 2015). flg22 induces IG production mainly by induction of MYB51, and secondarily of MYB122. Treatment with this MAMP additionally activates biosynthesis and PEN2-mediated metabolism of 4MO-I3M. In turn, generated end product(s) triggers in a positive feedback IG biosynthesis and activates, likely via unknown TFs, the biosynthesis of ICA and camalexin. Infection by P. cucumerina similarly switches on I3M biosynthesis via MYB TFs. However, it induces strongly indolic phytoalexin biosynthesis by activating TFs such as WRKY33 to induce expression of CYP79B2 independent of the three MYBs or the PEN2 pathway end product(s). Solid lines indicate single enzymatic steps, whereas dashed arrows denote several enzymatic steps. Dotted lines indicate positive activation by an effector. Different font size of MYB TFs indicates differential impact on the MAMP-/pathogen-triggered IG metabolism. Molecular Plant 2016 9, 682-695DOI: (10.1016/j.molp.2016.01.006) Copyright © 2016 The Author Terms and Conditions