Volume 5, Issue 1, Pages 115-130 (January 2012) Defense-Related Calcium Signaling Mutants Uncovered via a Quantitative High- Throughput Screen in Arabidopsis thaliana  Ranf Stefanie , Grimmer Julia , Pöschl Yvonne , Pecher Pascal , Chinchilla Delphine , Scheel Dierk , Lee Justin   Molecular Plant  Volume 5, Issue 1, Pages 115-130 (January 2012) DOI: 10.1093/mp/ssr064 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 MAMPs and DAMPs Induce Specific [Ca2+]cyt Elevations and Potentially Release of Vacuolar Ca2+ in Arabidopsis Seedlings. (A) [Ca2+]cyt elevations in Aeqcyt/Col-0 seedlings induced by the indicated MAMPs/DAMPs. Data are shown as L/Lmax-normalized values in the left graph and after conversion to [Ca2+]cyt in the right graph. (B) [Ca2+] elevations in Aeqvmd/C24 seedlings induced by the indicated MAMPs/DAMPs. Data are shown as L/Lmax-normalized values. To facilitate visualization, the lower part of the left graph is shown enlarged in the right graph. Arrows mark time of MAMP/DAMP application and data represent mean ± SD of at least three independent experiments (n ≥ 30) for both (A) and (B). Molecular Plant 2012 5, 115-130DOI: (10.1093/mp/ssr064) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Ca2+-Based Screening Reveals Novel fls2 Alleles. (A) Schematic illustration of the fls2 mutations. Mutations in the Aeqcyt (Col-0) background are marked with black and mutations in the Aeqvmd (C24) background with gray arrowheads. Open arrowheads mark SNPs between Aeqvmd/C24 and Col-0. fs, frame shift; splice, mutation of exon-intron border; -, nonsense mutation; SP, signal peptide; LRR, leucine-rich repeat domain; NT/CT, N/C-terminal LRR domain; TM, transmembrane domain; JM, juxtamembrane domain; S/T kinase, serine/threonine kinase domain (gray bar = catalytic loop, dashed lines = proton and ATP binding sites). (B) FLS2 protein accumulation was analyzed by immunoblot using anti-FLS2 antibodies (targeted against the KANSFREDRNEDREV epitope sequence corresponding to the FLS2 C-terminus, as indicated by the ‘Y’ symbol). Amido-black-staining of the Rubisco large subunit is used as an estimation of equal loading. Three independent experiments revealed identical results. (C) [Ca2+]cyt elevations in Aeqcyt fls2 mutant seedlings induced by 1 μM flg22 each compared to its respective wild-type control. Arrows mark time of flg22 application. Data represent mean of calibrated [Ca2+]cyt ± SD of at least four independent experiments (n ≥ 30). (D) [Ca2+] elevations in Aeqvmd fls2 mutant seedlings induced by 1 μM flg22 each compared to its respective wild-type control. Arrows mark time of flg22 application. L/Lmax-normalized data are shown as mean ± SD of at least six independent experiments (n ≥ 45). (E) Root growth inhibition (1 μM flg22) of different fls2 mutants (black bars) each compared to its respective wild-type control (shaded bars). Data are given as percent inhibition compared to untreated control (the raw data of the root length and corresponding seedling photos are shown in Supplemental Figure 2); mean ± SE of at least three independent experiments (n ≥ 45); * indicates statistically significant difference (p < 0.001). Molecular Plant 2012 5, 115-130DOI: (10.1093/mp/ssr064) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Ca2+-Based Screening Reveals Novel bak1 Alleles. (A) Schematic illustration of the bak1 mutations. Mutations in the Aeqcyt (Col-0) background are marked with black and mutations in the Aeqvmd (C24) background with gray arrowheads. Splice, mutation of exon–intron border; -, nonsense mutation; SP, signal peptide; LRR, leucine-rich repeat domain; NT, N-terminal LRR domain; PR, proline-rich domain; TM, transmembrane domain; JM, juxtamembrane domain; S/T kinase, serine/threonine kinase domain (gray bar = catalytic loop, dashed lines = proton and ATP binding sites). (B) BAK1 protein accumulation was analyzed by immunoblot using anti-BAK1 antibodies (directed against the DSTSQIENEYPSGPR sequence derived from the BAK1 C-terminus, as indicated by the ‘Y’ symbol). Amido-black-staining of the Rubisco large subunit suggests equal loading. Three independent experiments revealed identical results. (C, F) [Ca2+]cyt elevations in Aeqcyt bak1 mutant seedlings induced by (C) 1 μM flg22 and (F) 1 μM elf18 each compared to its respective wild-type control. Arrows mark time of MAMP application. Data represent mean of calibrated [Ca2+]cyt ± SD of at least three independent experiments (n ≥ 45). (D, G) [Ca2+] elevations in Aeqvmd bak1 mutant seedlings induced by (D) 1 μM flg22 and (G) 1 μM elf18 each compared to its respective wild-type control. Arrows mark time of MAMP application. L/Lmax-normalized data are shown as mean ± SD of at least five independent experiments (n ≥ 20). (E, H) Growth inhibition of different bak1 mutants (black bars) each compared to its respective wild-type control (shaded bars) was analyzed by (E) root length (1 μM flg22) or (H) fresh weight (100 nM elf18) examination. Data are given as percent inhibition compared to untreated control (the raw data and seedling photos are additionally shown in Supplemental Figure 3); mean ± SE of at least three independent experiments (n ≥ 24); * indicates statistically significant difference; ns, not significant (p < 0.001). Molecular Plant 2012 5, 115-130DOI: (10.1093/mp/ssr064) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 cce Mutants Show Reduced or Enhanced Responses to flg22 and elf18. (A, G) [Ca2+]cyt elevations in Aeqcyt cce mutant seedlings induced by (A) 1 μM flg22 and (G) 1 μM elf18 each compared to its respective wild-type control. Arrows mark time of MAMP application. Data represent mean of calibrated [Ca2+]cyt ± SD of at least three independent experiments (n ≥ 45). [Ca2+] peaks chosen for calculation (D, J) are marked with (1) or (2). (B, C, H, I) [Ca2+] elevations in Aeqvmd cce mutant seedlings induced by (B, C) 1 μM flg22 and (H, I) 1 μM elf18 each compared to its respective wild-type control. Arrows mark time of MAMP application. L/Lmax-normalized data are shown as mean ± SD of at least three independent experiments (n ≥ 26). [Ca2+] peaks chosen for calculation (D, J) are marked with (1) or (2). (D, J) Statistical analysis of MAMP-induced [Ca2+] elevations in cce mutants. Amplitudes of the [Ca2+] peaks ([Ca2+] max) or the overall sum of the [Ca2+] elevation ([Ca2+] sum) induced by (D) 1 μM flg22 or (J) 1 μM elf18 were calculated for each cce mutant line and are given as percent of the respective wild-type control. Data represent mean ± SD of at least three independent experiments (n ≥ 26). * indicates statistically significant difference; ns, not significant (p < 0.001). [Ca2+] peaks chosen for calculation are marked with (1) or (2) in (A–C) and (G–I). (E, K) Growth inhibition of different cce mutants (black bars) each compared to its respective wild-type control (shaded bars) was analyzed by (E) root length (1 μM flg22) or (K) fresh weight (100 nM elf18) examination. Data are given as percent inhibition compared to untreated control (the raw data are additionally shown in Supplemental Figure 4); mean ± SE of at least three independent experiments (n ≥ 24); * indicates statistically significant difference; ns, not significant (p < 0.001). (F) FLS2 and BAK1 protein accumulation was analyzed by immunoblot using anti-FLS2 and anti-BAK1 antibodies. Amido-black-staining of the Rubisco large subunit suggests equal loading. Three independent experiments revealed identical results. Molecular Plant 2012 5, 115-130DOI: (10.1093/mp/ssr064) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions