MYC YFP N PIF5 YFP C N-TIC TIC Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 Supplemental Figure. TIC interacts with MYC in the nucleus. Bimolecular fluorescence complementation assay using N-YFP fused with MYC or PIF5, and C-YFP fused with full-length TIC or N- terminus TIC. Proteins were transiently co-expressed in Nicotiana benthamiana, and YFP fluorescence was detected under a Leica confocal microscope. BiFC experiments were performed three times, and always same results were observed from each experiments.
MYC / PPA Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 Salk_877 (myc-) Salk_7 (myc-) MYC amplicon (A) MYC amplicon (B) Atg (MYC) A myc- myc- B Supplemental Figure. Identification of myc mutants. Two independent T-DNA inserted myc mutants were isolated, and named myc- (Salk_7) and myc- (Salk_877), respectively. (A) Quantitative RT-PCR of MYC expression relative to PPA in, myc-, and myc- was performed. The measurements of gene expression indicate a mean of three technical replicates, and error bars indicate standard deviation. The MYC expression level and standard deviation in myc- was below detectable level. (B) MYC full-length transcript level in, myc-, and myc-. Transcript level was determined by RT-PCR with primers for amplifying MYC amplicon (B), as displayed above.
MYC / PPA Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95.5..5..5..5. MYCox Supplemental Figure. MYC transcript levels in MYCox plants. Quantitative RT-PCR of MYC expression relative to PPA in and MYCox was performed. The measurements of gene expression indicate a mean of three technical replicates, and error bars indicate standard deviation.
log cfu/cm log cfu/cm log cfu/cm 7 A 5 7 5 Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 day myc- day tic- myc- tic- B day day 5 tic- myc- tic- myc- tic- myc- tic- myc- day day C t m t cm Supplemental Figure. Examined genotypes are not susceptible to Pst COR - (A) Disease symptoms of, myc-, tic-, and tic- myc- plants to the infection of Pst COR -. The picture was taken days after innoculating bacteria. (B) in vivo titers of P. syringae in, tic-, myc-, and tic- myc- plants on the day of spraying (day ) or days after infection (day ). Bacterial counts were expressed as log (cfu/cm ). Error bars indicate standard error.
Relative gene expression to PPA Relative gene expression to PPA Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 8 8 tic- tic- AOS OPR OPCL ACX ACX ACX5 JAR JMT AOS OPR OPCL ACX ACX ACX5 JAR JMT Supplemental Figure 5. JA biosynthetic gene expression is not altered in tic- relative to the wild type. Quantitative RT-PCR of JA biosynthetic gene expression relative to PPA in and tic- plants. Plants were grown under WLc for 7 days, and total RNA was extracted. The measurements of gene expression indicate a mean of three technical replicates, and error bars indicate standard deviation. 5
Root length (cm) Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 A 5 elf- - MeJA + MeJA B C JAZ5 fold change 5 5 5 TAT fold change 5 5 elf- elf- Supplemental Figure. elf- does not show JA-defective phenotypes. (A) Inhibition of root growth of -day-old, elf- seedlings on 5 M MeJA. The results are the mean of seedlings, and error bars indicate standard deviation (B) The fold change of JAZ5 (B) and TAT (C) expression by exogenous MeJA treatment relative to basal expression level. Plants were grown for 7 days, and treated with 5 M MeJA for hour (B) or for hours (C), repectively. The measurements of gene expression indicate a mean of three technical replicates, and error bars indicate standard deviation.
TIC / PPA MYC / PPA Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 A B.7..5.... tic-.5..5..5. myc- myc- MYCox Supplemental Figure 7. TIC and MYC do not participate in cross-regulation of respective transcript accumulation. (A) Quantitative RT-PCR of MYC expression relative to PPA in and tic- plants. (B) Quantitative RT-PCR of TIC expression relative to PPA in and myc-, myc- and MYCox plants. Plants were grown under WLc for 7 days, and total RNA was extracted. The measurements of gene expression indicate a mean of three technical replicates, and error bars indicate standard deviation. 7
MYC-myc / PPA Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95.5..5..5. 8 ZT (h) Supplemental Figure 8. MYC is over-expressed without oscillation in MYCox plants. Quantitative RT-PCR of MYC-myc expression relative to PPA in MYCox plants. Plants were grown under h light / h dark for 7 days. The measurements of gene expression indicate a mean of three technical replicates, and error bars indicate standard deviation. 8
Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 A ZT ZT MG - + - + MYC-myc H B MYCox Dc Rc FRc MYC-myc H Supplemental Figure 9. MYC protein is degraded by the proteasome. Immunoblot analysis for MYC-myc and histone H protein. (A) MYCox plants were grown under h light / h dark for 7 days. On 8 th day either at ZT or ZT, plants were treated with.8% DMSO or 8 M MG for hours. Total plant protein was extracted and MYC-myc and histone H protein were detected by immunoblot analysis. (B) MYCox plants were grown under continuous dark (Dc), continuous red (Rc), or continuous far-red (FRc) light for days, and total protein was extracted. MYC-myc protein level was determined by western-blot analysis. 9
log cfu/cm Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 tic- ZT ZT 8 ZT ZT 8 day day Supplemental Figure. Susceptibility to Pst COR - is similar regardless of genotypes and infection time of a day. and tic- plants were grown under short day conditions (8h light/h dark), and Pst COR - was sprayed to and tic- plants either at ZT (dawn) or at ZT8 (dusk), respectively. in vivo bacterial titer was determined on the day of spraying (day ) or days after infection (day ). Bacterial counts were expressed as log (cfu/cm ). Error bars indicate standard error.
MYC / PPA Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 8 ZT (h) Supplemental Figure. Circadian clock gates the JAinduced MYC expression. Quantitative RT-PCR of MYC expression relative to PPA in plants. Plants were grown under h light / h dark for 7 days, and treated with 5 M MeJA for hour at different times of a day with hours interval. The measurements of gene expression indicate a mean of three technical replicates, and error bars indicate standard deviation.
Supplemental Table Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 MYC MYC-myc JAZ5 LOX VSP TAT PR PDF. COI TIC AOS OPR OPCL ACX ACX ACX5 JAR JMT PPA qrt-pcr primer CTA AAC CAC GTC GAA GCA GAG AGA CAA AGA TCC TCG AGC TGG TTC TTG ATT TGG AGT TTC TCT AGA GGA TCC CGG AGG TGG CAG TCA AGT CTT CCT CGG AGA TTA GCT GGA ACA ACA ACA GCA ACA AGA ACA AAA TCA GGT TGA CCG GTT ACA ATC TCT GGC TTG GGA GAG GCT TCA CTG CTG GTG CAT ACG GCA TGG GCT GAC CAG AGT AAA AGA CCG TCG TTG GCG CTT TGA CCT AGA TGA TAC CCT CCT CTC TAG CCA ACG GTC ACT GAG TAT GAT GGG TTC AAT CAT GTG AGA GGC TCA AGG ATA TTC CTT GTC TAT CCT CAC CCA ATT CTC TGC CCC TAG AGC GAC CGC CTT CTG CGG GCC GGC AGG ACC TCG CAA TGA GAT GGC CTT GTT GAT AGC CAA AAC AGT TGT GCG AGA AGC CAA GT GTT GCA TGA TCC ATG TTT GG GGA AGT TCT CGA GAC AAG GAA TGT AAT CGG CCA TGT ATT CTA GCT CCT GGC AGC CCT GAG TTC TTC AGG ATT TAG ATC CGA TGG TGA AGG CTT CCT TAA GCA GAG GCT TTG CTT CCG TTT CGG TTA TCA ACC GTT GGC GAC GAG AGA TCC ACA ACC TCG CCA CCA AAA CAA CAA AAT CCT CTC TCC CGG ATC CGC AGG GTT CCC ACA TGT AGA TTG GTT TGT TCG TTG ACG ATA TTG GTG GAT GGA TTC ATA TTC GTG GTT GAT CGG TTA TCT GAT AAG CTG CTC CCA GTT TTC CTT ACG CAG GTC CTG AGG CCG AGC TCA GGA AAT TAG CTC AGT CAT AGC AAA ACA GCC CTT GAC AAC CGT ATG GTT CAA AAT GGA GAT ATT CTA CTA CCA AAC TGT TTC CTC TCC TTC AGA TAC CTG ATG ATC CGG TCT TTG AGA AAA GCA AAA GAG CAT TCC CCA ATG CAG ATC CAA GAA GCC TGG GAA TCT CGG TTA TTT CGA GTT TCT CCC TGT GGG GAG TGT TGT TGT AGA AAC CAA TGA CCT ATC GCG ATC TGA GGA GTT GGT CCC GGG AGG CGG AGC TCG CAG CAT AGT AAG GAG CGT TGA TAT CGG ATG ACG ATT CTT CGT GCA G GCT TGG TCG ACT ATC GGA ATG AGA G
MYC Cloning primer CGC GCC CGG GAT GAC TGA TTA CCG GCT ACA ACC CGC GCC TAG GAC CGA TTT TTG AAA TCA AAC TTG TIC MYC PIF5 Gateway cloning primer GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AGA AGG AGA TAG AAC CAT GAT AGA AAT AGA GAA (for N- TIC) GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA CTA ACA ATT CCT CTC AAA CTT (for fulllength GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA TCA TIC) TTT CGT CTC GCC AGC GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AGA AGG AGA TAG AAC CAT GAC TGA TTA CCG GCT A GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA CTA ACA ATT CCT CTC AAA CTT GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AAT GGA ACA AGT GTT TGC T GGG GAC CACT TTG TAC AAG AAA GCT GGG TAT CAG CCT ATT TTA CCC AT myc- myc- LBb. Genotyping primer ATT TTG CCG ATT TCG GAA CTC GAG CTG GTT CTT GAT TTG TGG TTT TTC TTG GTT TCG ATG TTT GGT ACA ACC GCT CGT AAC TAC GCT ATA TTC TGG CAA CCG