cpk28 is not involved in mediating meja-induced root ... · c inflorescence stem anatomy of wt, ......

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Supplemental Figure 1. CPK28 is not involved in mediating MeJA-induced root growth inhibition. A Primary root growth of WT, cpk28 and complementation line pCPK28:CPK28-YFP was monitored on 0.5 x MS plates over 20 days. Values indicate means ± SE, n = 12 per genotype/experiment, seen in 2 independent experiments. B Growth inhibition of primary roots by MeJA. Root length of WT, cpk28 and complementation line pCPK28:CPK28-YFP was measured 6 days after sowing on 0.5 x MS containing 1 % MeOH ( - ) or 20 µM MeJA ( + ). Bars indicate means ± SE, n = 12 per genotype/experiment, different letters indicate significant differences (1-Way-ANOVA, p < 0.05, Tukey’s test, seen in 2 independent experiments).

Supplemental Data. Matschi et al. Plant Cell (2015) 10.1105/tpc.15.00024

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Supplemental Figure 2. JA biosynthesis or JA perception is required for the cpk28 growth phenotype. A Plant morphology of WT, cpk28-1, aos, cpk28-1 x aos, coi1-16 and cpk28-1 x coi1-16 at day 47. Scale bar: 5 cm. B Plant height in WT and different single and double mutants at day 47 (means ± SE, Student’s t-test; significance level ** = P < 0.01, n = 10, seen in 3 independent experiments).


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Supplemental Figure 3. JA biosynthesis or JA perception is required for altered gene expression in cpk28 mutants. A-D Expression of JA-responsive marker genes PDF1.2, VSP2, PR3 and PR4 (A), JA signal transduction components JAZ1 and JAZ10 (B), NST1 (C) and GA3ox1 (D) in WT, cpk28-1, aos, cpk28-1 x aos, coi1-16 and cpk28-1 x coi1-16 (31 dag). Values represent means of three biological replicates with two pooled plants each ± SE (two technical replicates per biological replicate). Repetition of the experiment showed similar results (Student’s t-test; significance levels * = P < 0.05 and ** = P < 0.01 compared to WT).


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Supplemental Figure 4. DELLA abundance is not influencing the cpk28 phenotype. A Plant morphology of WT, cpk28, rga28, cpk28 x rga, gai and cpk28 x gai at 40 dag. Scale bar: 5 cm. B Rosette diameter in WT and different single and double mutants at day 42 (means ± SE, Student’s t-test; significance level ** = P < 0.01, n = 10, seen in 2 independent experiments). C RGA protein amount in WT, cpk28 and rga. Central rosette tissue of 31 day old plants was harvested and analyzed for RGA protein amount. Three plants were pooled per sample, small letters depict individual samples per genotype. Proteins were extracted and analyzed by SDS-gel electrophoresis and Western Blot with anti-RGA antibody. Equal protein amounts per sample are visualized by Ponceau S staining of the membrane.


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Supplemental Figure 5. CPK28 does not influence suppressed resistance to fungal infection in JA biosynthesis or signaling mutants. A-B Alternaria brassicicola infection assay. Leaves of 31-day-old WT, cpk28, aos, cpk28-1 x aos, coi1-16 and cpk28-1 x coi1-16 plants were drop-inoculated with spores of Alternaria brassicicola. (A) Disease symptoms were monitored ten days after inoculation. (B) Mean values of disease indices are presented ± SE for at least 12 replicates, asterisks represent significant differences from WT (Student’s t-test; significance levels * = P < 0.05, ** = P < 0.01 and *** = P < 0.001). Scale bar = 1 cm.


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Supplemental Figure 6. JA metabolite content in whole cpk28 plants is similar to wild type regardless of age or mechanical wounding. JA metabolite content measured in whole rosettes. Wounding of plants was carried out by squeezing 3 leaves per rosette with a pair of forceps. Whole rosettes were harvested before (control) or 1 hour after wounding (wounded) at 20 dag (LD20) and 27 dag (LD27) (values represent means ± SE, n = 6 per genotype/treatment). Extraction, purification, and quantification of OPDA, JA, JA-Ile and 12-OH-JA are described in Methods.


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Supplemental Figure 7. Harvesting schemes of JA metabolite measurements and tissue-specific qRT-PCR A Harvesting scheme for JA metabolite measurements in Supplemental Figure 6 and following experiments. B Adapted harvesting scheme for JA metabolite measurements (Figure 5), qRT-PCR analyses (Figure 6) and fungal growth quantification (Figure 7D). L = Leaf tissue, I = inner part of the rosette, including meristem, petioles and newly derived leaves.

Supplemental Data. Matschi et al. Plant Cell (2015) 10.1105/tpc.15-00024

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Supplemental Figure 8. Tissue-specific Alternaria brassicicola infection assay. A-B Leaves of the innermost rosette whorl of 31-day-old WT, cpk28 and aos plants were drop-inoculated with spores of Alternaria brassicicola. (A) Disease symptoms were monitored ten days after inoculation. (B) Mean values of disease indices are presented ± SE for at least 15 replicates, asterisks represent significant differences from WT (Student’s t-test; significance levels ** = P < 0.01). Scale bar = 1 cm.


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Supplemental Figure 9. Elevated ROS levels are not the cause of the JA-dependent growth phenotype in cpk28. A ROS assay following treatment with 200 nM flg22 in WT, cpk28, aos, cpk28-1 x aos, coi1-16 and cpk28-1 x coi1-16 (31 dag). Leaf discs were incubated for 16 hrs in water, treated with 200 nM flg22 and ROS production was determined in a luminol-based assay. Values represent means of total photon counts over 1 hour ± SE (n=8). B ROS assay following treatment with 200 nM flg22 in WT, cpk28, rbohd and cpk28 x rbohd (31 dag).


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C-D Plant morphology of WT, cpk28, rbohd and cpk28 x rbohd at day 35 (LD35, C) and day 47 (LD47, D). Scale bar: 5 cm. E Rosette diameter and plant height in WT and rbohd single and double mutants at day 47 (n = 12). F-I Inflorescence stem anatomy of WT, cpk28, rbohd and cpk28 x rbohd at 47 dag. Circles indicate xylem tissue of vascular bundles with fewer developed lignified xylem tracheary elements in cpk28. PF = lignified phloem fibers. Scale bar: 100 µm. (1-Way-ANOVA, Tukey’s post-test, P<0.05, different letters indicate significant differences, values represent means ± SE).


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Supplemental Figure 10. Environmental stress-induced calcium perturbations are not the cause of the JA-dependent growth phenotype in cpk28. A Plant morphology of WT, cpk28 and fou2 at day 42. Scale bar: 5 cm. B Rosette diameter and plant height in WT, cpk28 and fou2 at 42 dag. C Inflorescence stem anatomy of WT, cpk28 and fou2 at 47 dag. Circles indicate xylem tissue of vascular bundles with fewer developed lignified xylem tracheary elements in cpk28. PF = lignified phloem fibers. Scale bars: 100 µm. D Rosette diameter and plant height in WT, cpk28, tpc1-2 and cpk28 1 x tpc1-2 at 47 dag. (Statistical analysis: 1-Way-ANOVA, Tukey’s post-test, P<0.05, different letters indicate significant differences, values represent means ± SE, results in all panels were seen in at least 3 independent experiments).


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Supplemental Table 1. Genotyping primers used in this study.

name reaction sequence

CPK28 fwd CPK28 WT AAGGTTCCATGACATTGTTG

CPK28 rev CATTCGAAGTTCCTCAGGCGTTATG

GABI-KAT-LB T-DNA in cpk28 ATATTGACCATCATACTCATTGC

CPK28 rev CATTCGAAGTTCCTCAGGCGTTATG

AOS fwd AOS WT CTAACCGGAGGCTACCGTATC

AOS rev CGAGAAATTAACGGAGCTTCC

SALK-LB T-DNA in aos ATTTTGCCGATTTCGGAAC

AOS fwd CTAACCGGAGGCTACCGTATC

COI1-16 P2 COI1 WT GAACACAATTTAGTACTAAGGACGCATTCCCAA

COI1-16 P4 AACTAGTTGGGTTCTTTAAGGCTGCAGCTATTC

COI1-16 P2 coi1-16 (EMS

mutation)

GAACACAATTTAGTACTAAGGACGCATTCCCAA

COI1-16 P3 AACTAGTTGGGTTCTTTAAGGCTGCAGCTAACT

RGA fwd1 RGA WT CAGCTAAGCATCCGATTTGC

RGA rev TCACATAGAGAAGTCACATG

RGA fwd2 T-DNA in rga28 ATGAAGAGAGATCATCACC

SALK-LB ATTTTGCCGATTTCGGAAC

GAI fwd GAI WT ATGAAGAGAGATCATCATCAT

GAI rev ACCTTATCGATCGCACCAGGT

GAI T-DNA in gai ACCTTATCGATCGCACCAGGT

SAIL-LB TTTTCAGAAATGGATAAATAGCCTTGCTTC

RBOHD fwd RBOHD WT GCCTTTACACTCGCGACGCC

RBOHD rev CTTAAACCCTTTACTATTGTTGTTACTG

RBOHD rev T-DNA in rbohd CTTAAACCCTTTACTATTGTTGTTACTG


TPC1 fwd TPC1 WT ATATCGAAGAAAGCTCGGCTC

TPC1 rev GGGAAATAGAACCCGTGAGAG

TPC1 rev T-DNA in tpc1-2 GGGAAATAGAACCCGTGAGAG



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Supplemental Table 2. q(RT)-PCR primers used in this study.

name sequence

qRT-Actin2-F TCCCTCAGCACATTCCAGCAGAT

qRT-Actin2-R AACGATTCCTGGACCTGCCTCATC

qRT-NST1-F TCGAGCCTTGGGACATTCAAGAGA

qRT-NST1-R ACCAGTCGTTTTGTGGCGTTGTT

qRT-NST3-F GTGCCTCCAGGTTTCAGATTCC

qRT-NST3-R TCCTCTTGAATATCCCAAGGCTCA

qRT-GA3OX1-F ACCGTGCCACCGTTTCCTGG

qRT-GA3OX1-R TAACCCGAGCGCGGTGCAAC

qRT-PDF1.2-F CTTTCGACGCACCGGCAATG

qRT-PDF1.2-R TCCGCAAACCCCTGACCATG

qRT-CORI3-F GAGGCGGCCAAAGATGCT

qRT-CORI3-R CAGGGCATAGAGTGCAGAGGTA

qRT-VSP2-F TCCATCAACTACGCCAACTG

qRT-VSP2-R CGGTTTTGGAGTCGTATTGG

qRT-JAZ1-F AACTTGGCGAGCAAAGGCAC

qRT-JAZ1-R TGGATGAGGAACTTGGTTTGCGA

qRT-JAZ10-F ATGTCGAAAGCTACCATAGAACTCG

qRT-JAZ10-R GCCGATGAATCGGAATTGTTTCCAGTGG

qRT-AOS-F GAACCACCGGTTACGGCTCA

qRT-AOS-R ACTTTAAACGCCGCGTCGTG

qRT-OPR3-F GGTGTTAATGGCTCAAAGCTCGC

qRT-OPR3-R CTCTGTTTGCCCGTAGGCGT

qPCR-AbCUTAB1-F GCATGTCCGCTCACCAATATC

qPCR-AbCUTAB1-R GCCTGGGATCTTGGAATGC

qPCR-AtACTIN2-F CTTGCACCAAGCAGCATGAA

qPCR-AtACTIN2-R CCGATCCAGACACTGTACTTCCTT

SUPPLEMENTAL REFERENCES Dubiella, U., Seybold, H., Durian, G., Komander, E., Lassig, R., Witte, C.-P., Schulze, W.X. and Romeis, T. (2013) Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation. Proc Natl Acad Sci U S A, 110, 8744-8749. Willige, B.C., Ghosh, S., Nill, C., Zourelidou, M., Dohmann, E.M.N., Maier, A. and Schwechheimer, C. (2007) The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis. Plant Cell, 19, 1209-1220.


cpk28 is not involved in mediating meja-induced root ... · c inflorescence stem anatomy of wt, ......

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