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Figure S1. Nucleotide and deduced amino acid sequences of pepper CaAS1 cDNA encoding asparagine synthetase. The deduced amino acid sequence is given below the nucleotide sequence. The transcriptional start site is shown in bold type and the termination codon is marked by an asterisk (*). The black line and grey box indicate the AsnB and Asn synthase domains, respectively. A box indicates the polyadenylation sequence.

Figure S2. Comparisons of the deduced amino acid sequence of pepper asparagine synthetase, CaAS1, with asparagine synthetases from Triphysaria versicolor (accession no. AAD05035, Delavault et al., 1998), Elaeagnus umbellata (accession no. AAC16325, Kim et al., 1999), garden asparagus (Asparagus officinalis, accession no. CAA67889, Moyle et al., 1996), soybean (Glycine max, accession no. AAC09952, Yamagata et al., 1998), Arabidopsis (Arabidopsis thaliana ASN 1, accession no. NP_190318) and rice (Oryza sativa, accession no. BAD54377). Black and dotted lines indicate the AsnB and Asn synthase domains, respectively.

Figure S3. Expression of CaAS1 and defense-related genes in empty vector control (TRV:00) and silenced (TRV:CaAS1) pepper leaves. (a) RT-PCR analysis of CaAS1 expression in empty vector control (TRV:00) and silenced (TRV:CaAS1) leaves. H, Healthy leaves. (b) Real-time quantitative PCR analysis of the expression of pepper defense-related genes in empty vector control and silenced leaves at 0, 12, 24 and 36 h after inoculation with the avirulent (incompatible) strain Bv5-4a of Xanthomonas campestris pv.  vesicatoria.

Figure S4. Levels of different amino acids in wild-type (WT), empty vector control (TRV:00) and CaAS1-silenced pepper leaves that were not inoculated or inoculated with Xanthomonas campestris pv.  vesicatoria (Xcv). (a) Amino acid contents in wild-type, empty vector control (TRV:00) and silenced (TRV:CaAS1) leaves. (b) Changes in amino acid contents in empty vector control (TRV:00) and silenced (TRV:CaAS1) leaves at 0, 18 and 36 h after inoculation with the virulent (compatible) strain Ds1 and avirulent (incompatible) strain Bv5-4a of Xcv. Values are presented as means ± standard deviations. Different letters indicate significant differences, as analyzed by the LSD test (< 0.05). Asterisks indicate significant differences from empty vector control plants (Student’s t test, < 0.05). SER, serine; GLY, glycine; HIS, histidine; ARG, arginine; THR, threonine; ALA, alanine; PRO, proline; TYR, tyrosine; VAL, valine; MET, methionine; ILE, isoleucine; PHE, phenylalanine; TRP, tryptophan; and LYS, lysine.

Figure S5. Real-time quantitative PCR showing the expression of CaAS1 and Arabidopsis defense-related genes in wild-type (WT) and CaAS1-OX transgenic Arabidopsis plants (lines #1, #11 and #12). Values are presented as means ± standard deviations.

Figure S6. Distinct responses of wild-type and T-DNA insertion mutant Arabidopsis plants (asn1-1 and asn1-2) to infection with Pseudomonas syringae pv.  tomato (Pst) DC3000 and DC3000 (avrRpm1), and Hyaloperonospora arabidopsidis isolate Noco2. (a) Schematic representation of T-DNA insertion sites in asn1-1(SALK_144656) and asn1-2 (Salk_043167), and genomic structure of the Arabidopsis asparagine synthase 1 gene (ASN1). Exons and introns of ASN1 are represented as black and white boxes, respectively. (b) Bacterial growth in leaves of wild-type (WT) and T-DNA insertion mutant plants infiltrated with Pst DC3000 or DC3000 (avrRpm1) (105 cfu ml−1). (c) Quantification of sporangiophores produced on 50 cotyledons of wild-type (WT) and T-DNA insertion mutant plants inoculated with 5 × 104 conidiospores ml−1 of H.  arabidopsidis isolate Noco2. Production of sporangiophores was examined 6 days after inoculation. Different letters indicate significant differences, as analyzed by the LSD test (< 0.05) from three independent experiments.

Figure S7. Levels of different amino acids in wild-type (WT) and CaAS1-OX transgenic Arabidopsis leaves that were not inoculated or inoculated with Pseudomonas syringae pv.  tomato (Pst). (a) Amino acid contents in leaves of wild-type (WT) and transgenic plants (lines #1, #11 and #12). (b) Changes in amino acid contents in wild-type (WT) and transgenic plants (lines #11) at 0, 18 and 36 h after inoculation with Pst DC3000 or DC3000 (avrRpm1) (106 cfu ml−1). Different letters indicate significant differences, as analyzed by the LSD test (< 0.05). Values are presented as means ± standard deviations. Asterisks indicate significant differences from wild-type plants (Student’s t test, < 0.05). SER, serine; GLY, glycine; HIS, histidine; ARG, arginine; THR, threonine; ALA, alanine; PRO, proline; TYR, tyrosine; VAL, valine; MET, methionine; ILE, isoleucine; PHE, phenylalanine; TRP, tryptophan; LYS, lysine.

Appendix S1. Supplementary experimental procedures and results.

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