Transcript JAZ13-OE
0.6 * Root length ratio 0.5 * * * * * * * * #14 #15 #20 * 0.4 0.3 0.2 0.1 0 WT #3 #5 #7 #9 #11 #25 #30 35S::JAZ13 Figure S2. Overexpression of native (untagged) JAZ13 leads to decreased sensitivity to JAinduced root growth inhibition. T2 seedlings were grown under continuous light on ± 20 µM MeJA for 8 days. The root length ratio was calculated by dividing the average the root length of seedlings grown on MeJAcontaining medium by the average root length of seedlings of the same genotype grown in the absence of MeJA. Error bars indicate SE for at least 17 seedlings per genotype (n = 17 to 22). All lines were significantly different from WT (P < 0.005). WT JAZ13-OE Figure S3. JAZ13-OE leaves are deficient in anthocyanin accumulation in response to insect herbivory. WT and JAZ13-OE plants (line 26-3) challenged for 9 days with S. exigua neonate larvae. At the end of the feeding trial, a photograph was taken of representative plants of each genotype. Arrows denote anthocyanin accumulation in the midvein of the challenged WT plant, which was not observed in challenged JAZ13-OE plants. Similar results were obtained in three independent insect feeding trials. JAZ13 JAZ10 Empty Figure S4. JAZ13 homo- and heterodimerization assay in the yeast two-hybrid system. Yeast-two hybrid assay to assess the interaction of JAZ13 (DNA-binding domain (DB) fusion) with other JAZ proteins expressed as activation-domain (AD) fusions. MYC2 and JAZ10 (JAZ10.1 splice variant) were used as controls for positive interaction and JAZ-JAZ dimerization, respectively. a jaz8-V jaz8-Vashlovani C→A AflII 100bp jaz13-1 GK_193G07 b * ACTIN JAZ13 JAZ8 Figure S5 . Characterization of mutations in JAZ8 and JAZ13. (a) Gene structure JAZ8 and JAZ13 showing the position of mutations used in this study. The jaz8-V allele corresponds to a non-synonymous single nucleotide polymorphism (nsSNP) that creates a pre-mature stop codon in the TIFY/ZIM domain of JAZ8 from the Vashlovani-1 (Vash-1) accession. The CA SNP creates an AflII restriction site that was used for genotyping. jaz13-1 (GK_193G07) corresponds to a T-DNA insertion in exon 2 of JAZ13, which encodes the conserved NAFYXG motif within the ZIM domain. The position of the TDNA insertion is denoted by the inverted orange triangle. (b) RT-PCR analysis of JAZ8 and JAZ13 transcripts produced in the jaz13-1 and jaz8-V mutants. cDNA was synthesized from RNA obtained from wounded (1 h post treatment) and unwounded WT (Col-0) leaves, as well as wounded leaves from a jaz8-V jaz10-1 jaz13-1 (jaz8/10/13) triple mutant. Transcripts were amplified by PCR with the gene-specific primers listed in Table S2. ACTIN transcripts were used as a positive control. Primer locations for JAZ13 and JAZ8 are indicated by arrows in panel (a). Prior to gel electrophoresis, the JAZ8 amplicon was digested with restriction enzyme AflII to distinguish transcripts derived from the WT and jaz8-V alleles. Full-length jaz8-V transcripts accumulate in wounded leaves of the jaz8/10/13 mutant, as determined by the presence of two restriction fragments following digestion with AflII. RT-PCR analysis of JAZ13 transcripts resulted in amplification of a product (middle panel, asterisks) that is larger than the fully spliced JAZ13 transcript (lower band). Sequencing of PCR products showed that this band corresponds to an alternatively spliced form of JAZ13 in which an intron is retained, which is common for JAZ genes in Arabidopsis (Chung et al., 2010). a TCA (Ser58) > TAA (stop) 100 bp b JAZ8 jaz8-V * c EAR TIFY/ZIM Jas JAZ8Col 131 jaz8Vash 58 Figure S6. Sequence validation of a non-synonymous point mutation that creates a premature stop codon in the JAZ8 gene from accession Vash-1. (a) Schematic diagram of the JAZ8 gene and location of sequence polymorphism in Col-0 (TCA codon encoding Ser58) compared to Vash-1 (TAA stop codon). (b) PCR product (JAZ8-f3/r3 primers) derived from JAZ8 genomic DNA in Col-0 and Vash-1 was purified and directly sequenced with JAZ8-f3 primer. Asterisk indicates CA transversion in Vash-1 accession, which creates an pre-mature stop codon within the TIFY/ZIM domain. AflII recognition site (5’-CTTAAG-3’) is underlined. (c) Predicted effect of the jaz8-V mutation on the JAZ8 protein. A 6 Root length (cm) 5 4 3 B 2 B B C 1 0 Control + MeJA Figure S7. JA-mediated root growth inhibition in various jaz double mutants. Root growth assay comparing WT (Col-0) and the indicated jaz mutants grown for 7 days on MS plates supplemented (+MeJA) or not supplemented (Control) with 25 µm MeJA. Data show the mean ± SD of 19 to 24 seedlings per genotype. Capital letters denote statistical significance at P < 0.001 using ANOVA-TUKEY HSD statistical test.