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Supplementary Information
The Arabidopsis PEPR pathway couples local and systemic plant immunity
Annegret Ross, Kohji Yamada, Kei Hiruma, Misuzu Yamashita-Yamada, Xunli Lu, Yoshitaka Takano,
Kenichi Tsuda, and Yusuke Saijo
Content
Supplementary Methods and Materials
Supplementary Figures and Legends
(Please see Supplementary Tables S1, S2, and S4 in separate Excel sheets)
Supplementary Table S3
Supplementary Table S5
Supplementary References
Supplementary methods and materials
Plant transformation
The genomic DNA sequences of the PROPEP3 (At5g64905) loci including the inter-genic and coding
regions were subcloned into the binary vector pAM-PAT for expressing their C-terminal VENUS fusions,
and then transformed in Arabidopsis thaliana (Col-0) via Agrobacterium mediated transformation
(Clough & Bent, 1998).
Ion leakage analysis
Ion leakage measurement was conducted essentially as described earlier (Heidrich et al, 2011). Leaves of
4-week old plants were infiltrated with Pst DC3000 AvrRpm1 at 108 cfu/ml. Leaf discs were excised at 1,
3, 5, 6, 8, 10 and 22 h post infiltration, washed in water for 30 minutes, and then transferred to 2ml fresh
water. An aliquot of 60 µL was removed and then subjected to the measurement of the electrolyte
accumulation at every time point.
Microarray analysis
Total RNA was isolated with RNeasy Mini kit supplied with RNase-Free DNase (Qiagen) according to
the manufacturer's instructions. RNA quality was assessed with RNA Nanochips on a Bioanalyzer
(Agilent). Biotinylated cRNA was prepared according to a standard Ambion protocol from 1 μg total
RNA (MessageAmp II-Biotin Enhanced Kit; Ambion). After amplification and fragmentation, 12.5 μg
of cRNA was hybridized for 16 h at 45°C on GeneChip ATH1-121501 Genome Array. GeneChips were
washed and stained with Fluidics Script FS450-004 in the Affymetrix Fluidics Station 450 and scanned
using a GeneChip Scanner 3000 7G. The data were analyzed with Affymetrix GeneChip Operating
Software version 1.4 using Affymetrix default analysis settings and global scaling for signal
normalization. Probe signal values were subjected to the quantile normalization (Bolstad et al, 2003) and
summarization using the GeneChip robust multi-array average (GC-RMA) algorithm (Wu & Irizarry,
2004), and then the expression values of the genes were determined. The results were analyzed by the
following linear model using the lmFit function in the limma package in the R environment: log2
(expression level value)~ sample + replicate. The eBayes function in the limma package was used for
variance shrinkage in calculation of the p-values. The q-value function in the q-value package was used in
calculation of the Storey’s q-values (Storey & Tibshirani, 2003). All the microarray data obtained in this
study were submitted to Gene Expression Omnibus (Accession GSE40354).
For the gene ontology analysis, the genes selected were analyzed for the significance in the overrepresentation of the genes responsive to SA, MeJA, or ethylene by using Fisher’s exact test. The datasets
cross-referenced were Exp. ID AT-00110, Exp. ID AT-00113, and Exp. ID AT-00320 in Genevestigator
v3, which were also obtained in GeneChip ATH1-121501 Genome Array.
Statistical analysis for qRT-PCR experiments
The following models were fit to the Ct values using the lmer function in the lme4 package in the R
environment: Ctgr = Gg+Rr+egr (Figures 6B); Ctgtr = GTgt+Rr+egtr (Figures 2C, 3E, 5B, 6 and S7); Cttr =
Tt+Rr+etr (Figures 2A, 3A, 4A, 5A, 5C, 5D, S4A and S4B), where fixed factors; G, genotype factors; GT,
genotype:treatment interactions; T, treatment factors, and random factors; R, replicate; e, residual. The
mean estimates of the fixed factors were used as the modeled Ct values. The relative log2 expression
values were obtained by subtracting the Ct values of the genes from the Ct values of the At4g26410 gene
and compared using two-tailed t-tests. For the t-tests, the standard errors appropriate for the comparisons
were calculated using the variance and covariance values obtained from the model fittings.
Supplementary Figures and Legends
Figure S1. A role for the PEPR pathway in local immune responses. (A) qRT-PCR analysis in 10day-old WT and rsw3 seedlings treated with 1 µM Pep2 for the indicated times. Bars represent means and
standard errors of two biological replicates calculated by the mixed linear model. The vertical axis is the
log2 expression level relative to that of At4g26410. Statistical analysis (two-tailed t-tests) indicate not
significant differences from the Pep-treated wild-type plants (n.s.). (B) Leaves (n = ~30) of 4-week-old
plants were drop-inoculated with Colletotrichum higginsianum (Ch) path-29 (5 x 105 spores/ml). Lesion
sizes ±SD at 5 dpi are shown. For the pathogen inoculation assays student’s t test and BH method was
carried out to determine the significance of the difference between the mutant and wild-type plants in the
same treatment. Asterisk (*) indicate significant differences with q < 0.05; double asterisk (**), q < 0.01.
Figure S2: Transcriptome profiling in seedlings exposed to elf18 or Pep2 for 10 h. (A) The xaxis shows the log2 ratios of transcript levels in WT versus pepr1 pepr2 seedlings upon Pep2 (1 µM) for
10 h (q ≤ 0.05), and the y-axis shows the log2 ratios of transcript levels in WT versus efr seedlings upon
elf18 (1 µM) (q ≤ 0.05). The regression of this scatter plot is indicated by the bold line. Genes selectively
up-regulated upon Pep2 (> 1 [log2] for Pep2 and < 1 [log2] for elf18, q < 0.05) and those commonly upregulated upon both elicitors (> 3 [log2], q ≤ 0.05) are highlighted in pink (group A) or orange (group B),
respectively. (B) The genes of the 2 groups defined in (A) are cross-referenced to public database for their
expression responses to ACC (ET), MeJA or SA (Genevestigator v3). Each row represents the values of
biologically independent different datasets. The relative expression (in log2 ratios) is colored red for
induction and green for repression as illustrated in the fold change color bar.
Figure S3. Differences between Pep2 and flg22/elf18 in the induction of PDF1.2a encoding
defensin. (A and B) qRT-PCR analysis of PDF1.2a in 10-day-old seedlings treated with the indicated
elicitors at 1 µM for 24 h. Bars represent means and standard errors of at least four biological replicates
calculated by the mixed linear model. The vertical axis is the log2 expression level relative to that of
At4g26410. Asterisks indicate significant differences from Pep-treated (A) or untreated (B) wild-type
plants (*** = q < 0.001, two-tailed t-tests).
Figure S4: Ion leakage in pepr1 pepr2 leaves upon Pst DC3000 AvrRpm1 inoculation. Fourweek-old plant leaves were infiltrated with PstDC3000 AvrRpm1 (108 cfu/ml in 10 mM MgCl2),
detached, and then incubated in water for ion leakage measurement at 1, 3, 5, 6, 8, 10, and 22 h post
inoculation. Statistical analysis (two-tailed t-tests) indicate no significant differences between Pstinfiltrated or mock-infiltrated wild-type and pepr1 pepr2 plants.
Figure S5: Specificity of anti-PROPEP3 antibodies and functionality of PROPEP3-Venus.
(A) Synthesized peptides (3 µl) were blotted to the membranes at the indicated concentrations and then
probed with anti-PROPEP3 antibodies. (B) Nicotiana benthamiana leaves transiently expressing 19K,
Venus, or PROPEP3-Venus (from top left to right), and Arabidopsis WT (Col-0) and transgenic seedlings
expressing PROPEP3-Venus were subjected to the GFP-trap purification. The recovered proteins were
blotted onto the membrane and then probed with anti-GFP antibodies (left) and with anti-PROPEP3
antibodies (right). Synthesized Pep2 and Pep3 peptides were also blotted at the indicated concentrations
(right). (C) Root length of 8-day-old seedlings treated with a 1/100 dilution of the GFP-trap samples
tested in (B) for 5 days before the measurement. The Nb (-) samples used were prepared from Nb leaves
transiently expressing Venus. Student’s t test was carried out to determine the significance of the
differences between the PROPEP-Venus and control samples. Triple asterisk indicates a significant
difference at p < 0.001.
Table S3. Gene enrichment analysis in response to SA, MeJA and ethylene by Fisher's exact test.
Genes of selected classes from Figure 3A were analyzed for the significance in the over-representation of
the genes responsive to SA, MeJA and ethylene by using Fisher’s exact test. Bold p-values (< 0.001)
represent significant over-representation. The databases used were Exp. ID AT-00110, Exp. ID AT00113, and Exp. ID AT-00320 in Genevestigator v3.
Table S5. Primers used in this study.
Supplementary references
Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high
density oligonucleotide array data based on variance and bias. Bioinformatics 19: 185-193
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation
of Arabidopsis thaliana. Plant Journal 16: 735-743
Heidrich K, Wirthmueller L, Tasset C, Pouzet C, Deslandes L, Parker JE (2011) Arabidopsis EDS1
Connects Pathogen Effector Recognition to Cell Compartment-Specific Immune Responses. Science 334:
1401-1404
Storey JD, Tibshirani R (2003) Statistical significance for genomewide studies. P Natl Acad Sci USA 100:
9440-9445
Wu ZJ, Irizarry RA (2004) Preprocessing of oligonucleotide array data. Nat Biotechnol 22: 656-658