Volume 1, Issue 3, Pages 423-445 (May 2008) Activation of Defense Response Pathways by OGs and Flg22 Elicitors in Arabidopsis Seedlings  Denoux Carine , Galletti Roberta , Mammarella Nicole , Gopalan Suresh , Werck Danièle , De Lorenzo Giulia , Ferrari Simone , Ausubel Frederick M. , Dewdney Julia   Molecular Plant  Volume 1, Issue 3, Pages 423-445 (May 2008) DOI: 10.1093/mp/ssn019 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Transcript Profiles of Arabidopsis Seedlings Treated with OGs or Flg22. Ten-day-old seedlings were exposed to 50 μg ml−1 OGs or 1 μM Flg22 or an equal volume of water (control) for 1 or 3 h. Samples were hybridized to Affymetrix ATH1 GeneChips. (A) Numbers of differentially expressed genes (DEGs) that are induced or repressed by OGs or Flg22 at 1 or 3 h. Up-regulated genes are shown in orange and down-regulated genes in blue. (B) Correlation coefficients between treatments were calculated using DEGs identified with Rosetta Resolver. (C) SOM clustering of the differentially expressed genes identified by Rosetta Resolver. Gene up-regulation is indicated by orange, down-regulation by blue. Failure to meet criteria (P ≤ 0.01) for differential expression is indicated by grey. (O1) OGs 1 h treatment, (F1) Flg22 1 h treatment, (O3) OGs 3 h treatment, (F3) Flg22 3 h treatment. The color bar at the left demarcates individual clusters. Molecular Plant 2008 1, 423-445DOI: (10.1093/mp/ssn019) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Induction of Early-Response Genes Is Transient. Col-0 seedlings were elicited with OGs or Flg22 as for the expression profiling experiments, and harvested at 30 min or 1, 3, 6, 12, 24, or 48 h. The fold change relative to water-treated samples was determined by reverse-transcription–real-time PCR. Values shown are average and standard deviation of two independent experiments. Molecular Plant 2008 1, 423-445DOI: (10.1093/mp/ssn019) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 High Dose of OGs Does Not Recapitulate Response to Flg22. (A) Dose response analysis for OGs (O) and Flg22 (F). Gene induction was assayed at 1 h post treatment. Dose of OGs is shown in μg ml−1dose of Flg22 is shown as nM. (B) Extended time course analysis of the response to high and low doses of each elicitor. (C) Lifetime of the active elicitor in liquid media that contains seedlings. One or 3 h after addition of 50 μg ml−1 OGs or 1 μM Flg22 to medium containing seedlings, the medium was removed and added to fresh seedlings. These plants were then assayed for response. (D) Effect of repetitive elicitor addition. OGs (50 μg ml−1) or Flg22 (1 μM) were added to the medium every 30 min for 1 h (‘2×30’) or 3 h (‘6×30’). Seedlings harvested 1 h after the addition of a single dose of elicitor (‘1h’) were used as control. Expression was assayed by RT–qPCR. The fold change following elicitor treatment is relative to a water-treated sample. Values shown are average and standard deviation for two (A, C, D) or three (B) independent experiments. Molecular Plant 2008 1, 423-445DOI: (10.1093/mp/ssn019) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Early Signaling Is Differentially Activated by OGs and Flg22. (A) fls2 mutant plants are not impaired in recognition of OGs. Expression of CYP81F2 FAD-linked oxidase, and WRKY40 genes in 10-d-old Arabidopsis seedlings of ectoypes Col-0, Ler, Ws-0, or in the Ler fls2-24 mutant treated with water (H), 50 μg ml−1 oligogalacturonic acid (O), or 1 μM Flg22 (F) for 1 h. The fls2 mutant is in ecotype Landsberg erecta (Ler). Transcript levels were assayed by RT–PCR. The constitutively expressed ACTIN2 gene was included as a control for the amount of RNA used in each reaction. (B) Kinetics of MPK3 activation are different for OGs and Flg22. Activity of transiently expressed HA-tagged MPK3 in Arabidopsis mesophyll protoplasts was assayed using 32P gamma-ATP and myelin basic protein (MBP) as a substrate. Elicitations were performed with 1 μM Flg22 or 200 μg mL−1 OGs. Molecular Plant 2008 1, 423-445DOI: (10.1093/mp/ssn019) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Effect of Dose of OGs or Flg22 on Activation of Late Defense Responses. OGs and Flg22 were added to seedling medium to final concentrations of 50 μg ml−1 OGs, 1250 μg ml−1 OG, 10 nM Flg22, or 1000 nM Flg22, for 1, 3, 6, 12, or 24 h. Fold change was calculated from transcript levels in elicitor-treated seedlings compared to water-treated controls, as assayed by RT–qPCR. Values shown are average and standard deviation of two independent experiments. For LHCA6 values are ln(fold change). Molecular Plant 2008 1, 423-445DOI: (10.1093/mp/ssn019) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 OGs and Flg22 Differentially Induce Callose Deposition and PR1 Expression. (A–C) Seedlings (A) or leaves of 4-week-old plants (B,C) were treated with 200 μg ml−1 OGs,1 μM Flg22, or water by submergence (A, B) or infiltration (C). After 24 h, leaves were analyzed for GUS expression. (D–F) Seedlings (D) or leaves of 4-week-old plants (E,F) were treated with 200 μg ml−1 OGs, 1 μM Flg22, or water by submergence (D,E) or infiltration (F). At 18 h, leaves were stained with aniline blue for detection of callose, which was observed by fluorescence microscopy. Molecular Plant 2008 1, 423-445DOI: (10.1093/mp/ssn019) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 7 Flg22 Induction of PR1 Is Dependent on SA. Ten-day-old seedlings grown in liquid culture were treated with 100 μg/ml OGs or 1 μM Flg22 or an equal volume of water (control) and harvested 24 h after the addition of elicitor. Levels of PR1 transcript were determined by RT–qPCR, normalized to EIF4A1 and relative fold change calculated in comparison to control plants as described in Methods. Relative fold change is expressed as percent of change in wild-type plants treated with Flg22. Values shown are average and standard deviation of two independent experiments. Molecular Plant 2008 1, 423-445DOI: (10.1093/mp/ssn019) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions