Supporting Information Legends
Figure S1. Characterization of the cpk5 cpk6 cpk11 mutant. (a) Genotype analysis of the cpk5 cpk6 cpk11 TDNA insertion mutant. The CPK5, CPK6 and CPK11 genes were analyzed by PCR in wild-type and triple mutant
seedlings, using gene-specific primers flanking the left and the right borders of T-DNA insertion and genomic
DNA as a template. Levels of phosphorylated MAPKs (pMPK3 and pMPK6) in Col-0 and cpk5 cpk6 cpk11
seedlings treated with water, OGs or flg22 (b) and elicitor-infiltrated leaves (c). pMPK3 and pMPK6 were
detected by immunoblot analysis using a commercial antibody generated against the human homologs of
these MAPKs (α-p44/p42-ERK) (top panel). Levels of MPK3 and MPK6 total proteins were determined using
specific antibodies (bottom panel). The identity of individual MAP kinases, as determined by size, is indicated
by arrows. Experiments were repeated at least three times with similar results. (d) Chlorophyll release at the
indicated time points from Col-0 and cpk5 cpk6 cpk11 leaves. Data are the means of ten biological samples (±
SE). fw: fresh weight.
Figure S2. B. cinerea-induced expression of defense response genes in the cpk5 cpk6 cpk11 mutant. Wildtype and mutant leaves were inoculated with B. cinerea spores or potato dextrose broth (PDB; mock).
Transcript levels of the indicated genes were analyzed by qRT-PCR and shown as the mean of at least three
independent experiments (± SE; n = 4 in each experiment) normalized to UBQ5 expression and plotted relative
to expression in mock-treated wild type at 24 h. Asterisks indicate statistically significant differences according
to Student’s t test (*, P < 0.05; **, P < 0.01).
Figure S3. Basal and elicitor-induced expression of defense response genes in the cpk5 cpk6 cpk11 mutant.
(a) Analyses were performed in leaves from wild-type and cpk5 cpk6 cpk11 4-week-old plants by qRT-PCR and
transcript levels are shown as the mean of at least three independent experiments (± SE; n = 3 in each
experiment) normalized to UBQ5 expression. (b) Expression of the indicated defense response genes in Col-0
and cpk5 cpk6 cpk11 seedlings, analyzed by qRT-PCR after treatments with water, OGs or flg22 at the indicated
time point. Transcript levels are shown as the mean of at least three independent experiments (± SE; n = 20 in
each experiment) normalized to UBQ5 expression and plotted relative to expression in water-treated Col-0. In
a and b asterisks indicate statistically significant differences between mutant and Col-0, according to Student's
t test (* P < 0.05; ** P < 0.01).
Figure S4. Elicitor-induced MAPKs activation, induction of a subset of defense response genes and inhibition
of auxin-regulated gene expression by OGs are not affected in the ein2-5 mutant. (a) Levels of
phosphorylated MAPKs (pMPK3 and pMPK6) after treatments with water, OGs or elf18 in Col-0 and ein2-5
seedlings were determined by immunoblot analysis using a commercial antibody generated against the human
homologs of these MAPKs (α-p44/p42-ERK) (top panel). Levels of MPK3 and MPK6 total proteins were
determined using specific antibodies (bottom panel). The identity of individual MAP kinases, as determined by
size, is indicated by arrows. (b) Expression of the indicated defense response genes in Col-0 and ein2-5
seedlings, analyzed by qRT-PCR after treatments with water, OGs or elf18 at the indicated time points.
Transcript levels are shown as the mean of at least three independent experiments (± SE; n = 20 in each
experiment) normalized to UBQ5 expression and plotted relative to expression in water-treated Col-0. (c)
Expression of the indicated auxin-regulated genes in Col-0 and ein2-5 seedlings, treated for 1 hour with water,
IAA (1.5 μM), IAA+OGs or IAA+elf18 as indicated. Analyses were performed by qRT-PCR and transcript levels
are shown as the mean of at least three independent experiments (± SE; n = 20 in each experiment)
normalized to UBQ5 expression and plotted relative to expression in water treated Col-0. In b and c, asterisks
indicate statistically significant differences between mutant and wild-type (b) and between co-treatment with
IAA + elicitor and treatment with IAA alone (c) according to Student's t test (**, P < 0.01). All the experiments
were repeated three times with similar results.
Figure S5. OG-induced expression of late defense response genes is differentially affected in cpk5 cpk6
cpk11 mutant seedlings. Expression of the indicated late defense marker genes in wild-type and cpk5 cpk6
cpk11 seedlings at 3 hours after treatment with water, OGs or flg22 was analyzed by qRT-PCR. Transcript levels
are shown as the mean of at least three independent experiments (± SE; n = 20 in each experiment)
normalized to UBQ5 expression and plotted relative to expression in water-treated Col-0. Basal expression of
PAD3, normalized to UBQ5 expression, is shown in inset. Asterisks indicate statistically significant differences
between mutant and Col-0 seedlings, according to Student's t test (*, P < 0.05).
Figure S6. Elicitor-induced ROS production, callose deposition and inhibition of auxin-regulated gene
expression by OGs are not affected in the cpk5 cpk6 cpk11 mutant. (a) ROS production measured in relative
light units (RLUs) in 4-week-old Col-0 and cpk5 cpk6 cpk11 leaf discs after elicitation with water, OGs or flg22.
Results are average ± SE (n = 12). Similar results were obtained in five different experiments. (b) Wild-type and
cpk5 cpk6 cpk11 14-day-old seedlings were treated for 30 min with water, OGs or flg22 and the accumulation
of extracellular hydrogen peroxide in the incubation medium was determined by a xylenol orange based assay.
Results are means of three independent experiments (± SE; n = 40 in each experiment). (c) Leaves from
Arabidopsis wild-type and cpk5 cpk6 cpk11 plants were syringe-infiltrated with water, OGs or flg22. Twentyfour hours later leaves were excised and stained with aniline blue for callose visualization. This experiment was
repeated twice with similar results. (d) Expression of the indicated auxin-regulated genes in wild-type and cpk5
cpk6 cpk11 seedlings, treated for 1 hour with water, IAA (1.5 μM) or IAA+OGs, as indicated, was analyzed as
described in Figure S4c. Asterisks indicate statistically significant differences between co-treatment with IAA +
elicitor and treatment with IAA alone, according to Student's t test (*, P < 0.05; **, P < 0.01).
Figure S7. Basal resistance and OG-induced protection against B. cinerea is not impaired in cpk5, cpk6 and
cpk11 single mutants. Col-0 and the indicated cpk mutant plants were sprayed with OGs or water and, after 24
hours, leaves were inoculated with B. cinerea spores. Lesion areas were measured at 48 hpi. Results are
average ± SE (n = 20). Different letters above bars indicate statistically significant differences (P < 0.05)
between samples, as determined by ANOVA with Tukey-HSD test. The experiment was repeated three times
with similar results.
Supporting Table S1. Primers used in this work.