Sup. Fig.S1 Genotype and plant height analysis of the F1 progenies of TTxWW
a) Schematic diagram showing the genomic organization of tryptophan decarboxylase genes TDC-1 and TDC-3 and the location of the T-DNA insertion site. The orientation of the right border (RB)in the T-DNA is indicated. The solid box denotes the GUS gene, and the triangle denotes four copies of 35S enhancers. The set of primers a2 and b2, located on either side of the T-DNA as indicated by arrows, were used forprogeny genotyping of mutant M47286. Bar=1 kb.
b) Genotyping of M47286 TT (4 plants) × WW (wild-type) F1 progenies with a2/b2 or RB/b2 primers. All 12 plants are heterozygous genotype plants. No wild type segregation was observed.
c) The height of 12 plants of wild type and M47286 TT (4 plants) × WW (wild-type) F1 progenies.
Sup. Fig.S2 Expression of TDC-1 and TDC-3 in various genotypes of M47286 by quantitative RT-PCR
The cDNA samples fromthe 70-day-old leaves of wild type (67) and various genotypes of mutant M47286 were analyzed by quantitative RT-PCR. RNA levelswere quantified and normalized to the level of rRNA. The highest mRNAlevel in TT lines was assigned a value of 100, and mRNA levels of other sampleswere calculated relative to this value. Error bars indicate the SE for threereplicate experiments.
Sup. Fig.S3 RT-PCR analysis of the expression of TDC-related genes in TNG67 and in various genotypes of M47286
a)The cDNA samples fromthe 70-day-old leaves of wild type (67) and various genotypes of mutant M47286 were used to detect the expression of TDC-1, TDC-2, TDC-3, AAK50420, AAT44136, AAM01094 and AK065830 genes by RT-PCR assay.The expected sizes of PCR products from each gene were indicated. The expression of the actin gene was used as an RNA quality control. Reaction with 25 PCR cycles was performed.
b)PCR using genomic DNA for those genes not detected in RT-PCR were performed to demonstrate the primers used in the RT-PCR reaction were correct and appropriate.
Sup. Fig.S4 Phylogenetic analysis of rice aromatic amino acid decarboxylase AADC and the enzymatic activity of TDC-1 and TDC-3
a) Phylogenetic tree showing the relationship among 7 rice TDC-like or TYDC-like enzymes. The tree was created with the ClustalW program by comparing full-length amino acid sequences. Bar = 0.1 amino acid substitutions per site.
b) Metabolic pathway from tryptophan to serotonin in plants. Tryptophan can be catalyzed by TDC and T5H to form serotonin, which can be further catalyzed to form serotonin dimer upon exposure to UV light.
c) Enzymatic activity assay of TDC-1 and TDC-3. Purified recombinantTDC-1 (square) or TDC-3 (triangle) from E. coli (6 g) were incubated with 10 mM L-tryptophan at 30°C, and changes in the concentrations of tryptamine (■ and ▲) and L-tryptophan(□ and△)at various time intervals were determined by HPLC. The data represent the mean values of three independent assays.
Sup. Fig.S5 Southern blot analysis to identify the T-DNA insertion events in TDC-1 and TDC-3 transgenic rice
Genomic DNA (15 g) from wild type (67)and different Ubi:TDC-1 and Ubi-TDC-3 transgenic lines as indicatedin the figures was digested with Xba I and hybridized with a GUS DNA probe. Molecular size markersin kb are shown on the left side of the gel.
Sup. Fig.S6Serotonin and serotonin dimer contents in the rice blast infected leaves
The contents of serotonin (b) and serotonin dimer (c) in the healthy (67)and rice blast-infected leaves (a) were analyzed using the methods described in the Materials and Methodssection.